Nitrogen-regulated sugar sensing gene and protein and modulation thereof

ABSTRACT

The present invention relates to a nitrogen-regulated GATA transcription factor gene required for sugar sensing and the modulation of the expression of this gene to modulate a characteristic in a plant. The GATA transcription factor of the present invention is involved in regulating sugar sensing in plants and its expression is influenced by nitrogen status. Increased expression of this or substantially similar genes can produce plants with improved nitrogen utilization and increased yield.

This is a divisional of U.S. application Ser. No. 11/331,199, filed Jan.13, 2006, pending, which claims the benefit of U.S. ProvisionalApplication No. 60/643,575, filed Jan. 14, 2005.

FIELD OF THE INVENTION

The present invention relates to methods of modulating agronomic traitsin plants by modulating the expression of a GATA transcription factor inthe plant cells. In particular the present invention relates to methodsof improving nitrogen utilization in plants. The present invention alsopertains to nucleic acid molecules isolated from Arabidopsis thalianacomprising nucleotide sequences that encode proteins that are sugarsensing and, ultimately, can modulate nitrogen uptake and overall carbonmetabolism.

BACKGROUND OF THE INVENTION

Improvement of the agronomic characteristics of crop plants has beenongoing since the beginning of agriculture. Most of the land suitablefor crop production is currently being used. As human populationscontinue to increase, improved crop varieties will be required toadequately provide our food and feed (Trewavas (2001) Plant Physiol.125: 174-179). To avoid catastrophic famines and malnutrition, futurecrop cultivars will need to have improved yields with equivalent farminputs. These cultivars will need to more effectively withstand adverseconditions such as drought, soil salinity or disease, which will beespecially important as marginal lands are brought into cultivation.Finally, we will need cultivars with altered nutrient composition toenhance human and animal nutrition, and to enable more efficient foodand feed processing. For all these traits, identification of the genescontrolling phenotypic expression of traits of interest will be crucialin accelerating development of superior crop germplasm by conventionalor transgenic means.

A number of highly-efficient approaches are available to assistidentification of genes playing key roles in expression ofagronomically-important traits. These include genetics, genomics,bioinformatics, and functional genomics. Genetics is the scientificstudy of the mechanisms of inheritance. By identifying mutations thatalter the pathway or response of interest, classical (or forward)genetics can help to identify the genes involved in these pathways orresponses. For example, a mutant with enhanced susceptibility to diseasemay identify an important component of the plant signal transductionpathway leading from pathogen recognition to disease resistance.Genetics is also the central component in improvement of germplasm bybreeding. Through molecular and phenotypic analysis of genetic crosses,loci controlling traits of interest can be mapped and followed insubsequent generations. Knowledge of the genes underlying phenotypicvariation between crop accessions can enable development of markers thatgreatly increase efficiency of the germplasm improvement process, aswell as open avenues for discovery of additional superior alleles.

Genomics is the system-level study of an organism's genome, includinggenes and corresponding gene products—RNA and proteins. At a firstlevel, genomic approaches have provided large datasets of sequenceinformation from diverse plant species, including full-length andpartial cDNA sequences, and the complete genomic sequence of a modelplant species, Arabidopsis thaliana. Recently, the first draft sequenceof a crop plant's genome, that of rice (Oryza sativa), has also becomeavailable. Availability of a whole genome sequence makes possible thedevelopment of tools for system-level study of other molecularcomplements, such as arrays and chips for use in determining thecomplement of expressed genes in an organism under specific conditions.Such data can be used as a first indication of the potential for certaingenes to play key roles in expression of different plant phenotypes.

Bioinformatics approaches interface directly with first-level genomicdatasets in allowing for processing to uncover sequences of interest byannotative or other means. Using, for example, similarity searches,alignments and phylogenetic analyses, bioinformatics can often identifyhomologs of a gene product of interest. Very similar homologs (eg. >−90%amino acid identity over the entire length of the protein) are verylikely orthologs, i.e. share the same function in different organisms.

Functional genomics can be defined as the assignment of function togenes and their products. Functional genomics draws from genetics,genomics and bioinformatics to derive a path toward identifying genesimportant in a particular pathway or response of interest. Expressionanalysis, for example, uses high density DNA microarrays (often derivedfrom genomic-scale organismal sequencing) to monitor the mRNA expressionof thousands of genes in a single experiment. Experimental treatmentscan include those eliciting a response of interest, such as the diseaseresistance response in plants infected with a pathogen. To giveadditional examples of the use of microarrays, mRNA expression levelscan be monitored in distinct tissues over a developmental time course,or in mutants affected in a response of interest. Proteomics can alsohelp to assign function, by assaying the expression andpost-translational modifications of hundreds of proteins in a singleexperiment.

Proteomics approaches are in many cases analogous to the approachestaken for monitoring mRNA expression in microarray experiments.Protein-protein interactions can also help to assign proteins to a givenpathway or response, by identifying proteins that interact with knowncomponents of the pathway or response. For functional genomics,protein-protein interactions are often studied using large-scale yeasttwo-hybrid assays. Another approach to assigning gene function is toexpress the corresponding protein in a heterologous host, for examplethe bacterium Escherichia coli, followed by purification and enzymaticassays.

Demonstration of the ability of a gene-of-interest to control a giventrait may be derived, for example, from experimental testing in plantspecies of interest. The generation and analysis of plants transgenicfor a gene of interest can be used for plant functional genomics, withseveral advantages. The gene can often be both overexpressed andunderexpressed (“knocked out”), thereby increasing the chances ofobserving a phenotype linking the gene to a pathway or response ofinterest. Two aspects of transgenic functional genomics help lend a highlevel of confidence to functional assignment by this approach. First,phenotypic observations are carried out in the context of the livingplant. Second, the range of phenotypes observed can be checked andcorrelated with observed expression levels of the introduced transgene.Transgenic functional genomics is especially valuable in improvedcultivar development. Only genes that function in a pathway or responseof interest, and that in addition are able to confer a desiredtrait-based phenotype, are promoted as candidate genes for cropimprovement efforts. In some cases, transgenic lines developed forfunctional genomics studies can be directly utilized in initial stagesof product development.

Another approach towards plant functional genomics involves firstidentifying plant lines with mutations in specific genes of interest,followed by phenotypic evaluation of the consequences of such geneknockouts on the trait understudy. Such an approach reveals genesessential for expression of specific traits.

Genes identified through functional genomics can be directly employed inefforts towards germplasm improvement by transgenic means, as describedabove, or used to develop markers for identification of tracking ofalleles-of-interest in mapping and breeding populations. Knowledge ofsuch genes may also enable construction of superior alleles non-existentin nature, by any of a number of molecular methods.

Rapid increases in yield over the last 80 years in row crops have beendue in roughly equal measure to improved genetics and improved agronomicpractices. In particular, in a crop like maize, the combination of highyielding hybrids and the use of large amounts of nitrogen fertilizerhave under ideal conditions allowed for yields of greater than 440bu/acre. However, the use of large amounts of nitrogen fertilizer hasnegative side-effects primarily around increasing cost of this input tothe farmer and cost to the environment since nitrate pollution is amajor problem in many agricultural areas contributing significantly tothe degradation of both fresh water and marine environments. Developingcrop genetics that use nitrogen more efficiently through anunderstanding of the role of genotype on nitrogen use would be highlyadvantageous in reducing producer input costs as well as environmentalload. This is particularly important for a crop like corn which is grownusing a high level of nitrogen fertilizer.

Nitrogen use efficiency can be defined in several ways, although thesimplest is yield/N supplied. There are two stages in this process:first, the amount of available nitrogen that is taken up, stored andassimilated into amino acids and other important nitrogenous compounds;second, the proportion of nitrogen that is partitioned to the seed,resulting in final yield. A variety of field studies have been performedon various agriculturally-important crops to study this problem (LawlorD W et al 2001 in Lea P J, Morot-Gaudry J F, eds. Plant Nitrogen.Berlin: Springer-Verlag 343-367; Lafifte H R and Edmeades G O 1994 FieldCrops Res 39, 15-25; Lawlor D W 2002 J Exp Bot. 53, 773-87; Moll R H etal 1982 Agron J 74, 562-564). These experiments have demonstrated thatthere is a genetic component to nitrogen use efficiency, but have notproved satisfactory in determining which genes are important for thisprocess. In addition, corn breeders have generally not targeted themaintenance of yield under limiting nitrogen fertilizer. These types offield experiments on nitrogen use are difficult for a variety of reasonsincluding a lack of uniformity of accessible nitrogen in a test field orbetween field sites under any treatment regime and the interplay ofother environmental factors that make experiments difficult tointerpret.

Therefore, although there is experimental evidence for genetic variationfor this trait, it is difficult to make any conclusions from theseexperiments on what causes this variation. It should be feasible and iscertainly important to develop methods to study this trait under fieldconditions in crop plants. However, significant progress towardidentifying, understanding and manipulating important traits can be madethrough the use of a model system like Arabidopsis. At the very least,these experiments will give important clues about potential target genesto evaluate in important field crops. In addition, there are alsoconsiderable genetic and genomic resources available to study rice andthis species will also be used for some of the proposed experiments as aspecies more similar to corn than is Arabidopsis.

Nitrate is the major form of available nitrogen in the field and thereis an extensive body of literature on genes involved in nitrate uptakeand reduction (Forde B G 2000 Biochimica et Biophysica Acta 1465,219-235; Howift S M and Udvardi M K 2000 Biochimica et Biophysica Acta1465, 152-170; Stitt M et al 2002 J Exp Bot. 53, 959-70) as well as ongenes involved in other aspects of nitrogen metabolism (Lea P J,Morot-Gaudry J F, eds. 2001 Plant Nitrogen. Berlin; Springer-Verlag;Morot-Gaudry J F 2001 Nitrogen assimilation by plants Science PublishersInc. NH, US). Also, it is clear that the availability of carbonmetabolites is crucial for the efficient use of field nitrate and thereis good experimental evidence for a linkage between carbon and nitrogenmetabolism (Coruzzi G M and Zhou L 2001 Curr Opin Plant Biol. 4,247-53). In addition, some experiments suggest that GS and GOGAT areinvolved in remobilizing N from senescing organs to the sink organ(Brouquisse R et al 2001 in Lea P J, Morot-Gaudry J F, eds. PlantNitrogen. Berlin: Springer-Verlag 275-293; Yamaya T et al 2002 J ExpBot. 53, 917-925). However, most aspects of the regulation of thesegenes are still unclear and there is still no notion of how thisregulation affects nitrogen use efficiency.

Plants can sense levels of carbon and nitrogen metabolites andaccordingly adjust growth and development. The perception mechanisms arecomplex regulatory networks that control gene expression to accommodateconstant changes of nutrient-dependent cellular activities. Possessionof a sugar-sensing mechanism enables plants to turn off photosynthesiswhen C-skeletons are abundant. The N-sensing mechanism enables plants toturn off nitrate uptake and reduction when levels of reduced or organicN are high (Coruzzi, G. M. & Zhou, L. (2001) Curr Opin Plant Biol. 4,247-53).

Multiple sugar signal transduction pathways exist in plants. Glucose hasemerged as a key regulator of many vital processes in photosyntheticplants such as in photosynthesis and in carbon and nitrogen metabolism(Roland, F., Moore, B. & Sheen, J. (2002) Plant Cell S185-S205).Hexokinases (HXK) are an important control point for glucose metabolism.They not only catalyze the phosphorylation of glucose but also functionas a glucose sensor to interrelate nutrient, light and hormone signalingnetworks for controlling growth and development in response to thechanging environment (Jang, J., Leon, P; Zhou, L. & Sheen, J. (1997)Plant Cell 9, 5-19; Dai, N., Schaffer, A., Petreikov, M., Shahak, Y.,Giller, Y., Ratner, K., Levine, A. & Granot, D. (1999) Plant Cell 11,1253-1266; Moore, B., Zhou, L., Rolland, F., Hall, Q., Cheng, W., Liu,Y., Hwang, I., Jones, T. & Sheen, J. (2003) Science 300, 332-336). Inother organisms it has been shown that hexose transport molecules alsoserve as sugar sensors.

Multiple N signals and sensing pathways exist as well in plants. Plantshave mechanisms to sense nitrate, the major form of nitrogen fertilizer,as a signal for inorganic N status as well as to sense metabolitesderived from nitrate as signals for reduced or organic N status. Nitratereductase (NR) and nitrite reductase (NiR) are the first two enzymes inthe nitrate reduction process and their expression can be stimulated bythe presence of nitrate and modulated by other physiological factorsincluding some nitrogenous compounds, sucrose, light and hormone (Forde,B. G. (2000) Biochimica et Biophysica Acta 1465, 219-235; Howitt, S. M.& Udvardi, M. K. (2000) Biochimica et Biophysica Acta 1465, 152-170;Stift, M., Müller, M., Maft, M., Gibon, Y., Carilio, P., Morcuende, R.,Scheible, W. & Krapp, A. (2002) J Exp Bot. 53, 959-970; Lea, P. J. &Morot-Gaudry, J. F. eds. 2001 Plant Nitrogen. Berlin: Springer-Verlag;Morot-Gaudry J F 2001 Nitrogen assimilation by plants Science PublishersInc. NH, US).

It is clear that carbon and nitrogen metabolism is closely linked andtightly regulated (Coruzzi, G. & Bush, D. R. (2001) Plant Physiol 125,61-64). The availability of carbon metabolites is crucial for efficientnitrate utilization and the nitrogen status is very sensitive tophotosynthesis. Despite increased knowledge of structural genes involvedin carbon and nitrogen metabolism, trans-acting factors involved intranscriptional regulation of C/N gene expression have not beencharacterized.

GATA transcription factors are a group of transcriptional regulatorsbroadly distributed in eukaryotes. The GATA DNA binding domain normallyrecognizes the consensus sequence WGATAR (W=T or A; R=G or A) (Lowry, J.& Atchley, W. (2000) J Mol Evol 50, 103-115). GATA motifs have beenidentified in the regulatory regions of many light responsive genes(Arguello-Astorga, G & Herrera-Estrella, L. (1998) Annu Rev PlantPhysiol Plant Mol Biol 49, 525-555), including many genes involved in orrelating to photosynthesis such as the RBCS, CAB (chlorophyll A/Bbinding protein) and GAP (glyceraldehyde-3-phosphate dehydrogenase)(Terzaghi, W. B. & Cashmore, A. R. (1995) Annu Rev Plant Physiol PlantMol Biol 46, 445-474; Koch, K. E. (1996) Carbohydrate-modulated geneexpression in plants. Annu Rev Plant Physiol Plant Mol Biol 47, 509-540;Jeong, M. J. & Shih, M. C. (2003) Biochem Biophys Res Commun 300,555-562) as well as genes involved in nitrate assimilation such asnitrate reductase, nitrite reductase, and Gln synthetase (Jarai, G.,Truong, H., Daniel-Vedele, F. & Marzluf, G. (1992) Curr Genet. 21,37-41; Rastogi, R., Bate, N., Sivasankar, S & Rothstein, S. (1997) PlantMol Biol. 34, 465-76; Oliveira, I. C. & Coruzzi, G. M. (1999) PlantPhysiol 121, 301-309). Some known trans-acting regulatory proteins thatglobally regulate genes in N metabolism are GATA transcription factorgenes. In yeast, four global nitrogen regulatory factors GLN3, NIL1,NIL2 and DAL80 are DNA-binding proteins that contain a single GATA zincfinger, recognizing the consensus motif GATA (Hofman-Bang, J. (1999) MolBiotech 12, 35-73). In fungi, Neurospora crassa NIT2 (Tao Y and MarzlufG A 1999 Curr Genet. 36, 153-158) and Aspergillus nidulans AREA (CaddickM X Arst H N Jr Taylor L H Johnson R I Brownlee A G 1986 Cloning of theregulatory gene areA mediating nitrogen metabolite repression inAspergillus nidulans. EMBO J 5, 1087-1090) are GATA transcription factorgenes.

In plants, the in vivo function of GATA factors remains very poorlydefined, with the Arabidopsis genome having 30 GATA members (Riechmann,J. L., Heard, J., Martin, G., Reuber, L., Jiang, C., Keddie, J., Adam,L., Pineda, O., Ratcliffe, O. J., Samaha, R. R., Creelman, R., Pilgrim,M., Broun, P., Zhang, J. Z., Ghandehari, D., Sherman, B. K. & Yu, G.(2000) Science 290, 2105-2110; Reyes, J. C., Muro-Pastor, M. I. &Florencio, F. J. (2004) Plant Physiol. 134, 1718-1732).

SUMMARY OF THE INVENTION

In the attempt to understand the biological function of members of theGATA transcription factor gene family, a GATA transcription factor genewas studied to understand its role in the regulation of carbon andnitrogen metabolism. The inventors have determined that the expressionof the At5g56860 gene is influenced by the nitrogen status and itsexpression regulates the expression of genes controlling carbonmetabolism as well as genes involved in other biological processes.Loss-of-function mutant plants in the At5g56860 gene resulted in reducedchlorophyll level and these plants were hypersensitive to exogenousglucose. In contrast, gain-of-function transgenic plants were lesssensitive to exogenous glucose.

Sugars are central regulators of many vital processes in photosyntheticplants, such as photosynthesis and carbon and nitrogen metabolism. Thisregulation is achieved by regulating gene expression to either activateor repress genes involved. The mechanisms by which sugars control geneexpression are not understood well. The GATA transcription factordisclosed here is involved in regulating sugar sensing and theexpression of the factor itself is influenced by the change of the Nstatus. Increased expression of this gene can produce plants withincreased yield, particularly as the manipulation of sugar signalingpathways can lead to increased photosynthesis and increased nitrogenassimilation and alter source-sink relationships in seeds, tubes, rootsand other storage organs.

Accordingly, the present invention relates to a method of modulating acharacteristic in a plant or plant cell comprising modulating expressionof a GATA transcription factor gene in the plant or plant cell. In anembodiment of the invention, the expression of the GATA transcriptionfactor gene is modulated by administering, to the cell, an effectiveamount of an agent that can modulate the expression levels of a GATAtranscription factor gene in the plant cell. In a further embodiment ofthe invention, the agent enhances the expression levels of a GATAtranscription factor gene in the plant cell.

The characteristic to be modulated in the plant may be any agronomictrait of interest. In an embodiment of the invention, the characteristicis any that is affected by nitrogen, carbon and/or sulfur metabolism,biosynthesis of lipids, perception of nutrients, nutritional adaptation,electron transport and/or membrane associated energy conservation. In afurther embodiment of the invention, the characteristic is selected fromone or more of nitrogen utilization, yield, cell growth, reproduction,photosynthesis, nitrogen assimilation, disease resistance,differentiation, signal transduction, gene regulation, abiotic stresstolerance and nutritional composition. In a still further embodiment ofthe invention the modulated characteristic is an increase or improvementin one or more of nitrogen utilization, yield, cell growth,reproduction, photosynthesis, nitrogen assimilation, disease resistance,differentiation, signal transduction, gene regulation abiotic stresstolerance and nutritional composition.

In a particular embodiment, the present invention relates to a method ofimproving nitrogen utilization in a plant or plant cell comprisingenhancing expression of a GATA transcription factor gene in the plant orplant cell. Improving nitrogen utilization in a plant will allow forreduce amounts of nitrogen fertilizer to applied to the plant with aconcomitant reduction in costs to the farmer and cost to the environmentsince nitrate pollution is a major problem in many agricultural areascontributing significantly to the degradation of both fresh water andmarine environments.

The plant or plant cell may be from any plant wherein one wishes tomodulate a characteristic. In an embodiment of the invention, the plantcell is a dicot, a gymnosperm or a monocot. In one embodiment, the dicotis selected from the group consisting of soybean, tobacco or cotton. Ina further embodiment of the invention, the monocot is selected frommaize, wheat, barley, oats, rye, millet, sorghum, triticale, secale,einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum sp. andteosite.

In an embodiment of the invention, the agent that enhances theexpression levels of a GATA transcription factor gene in the plant cellcomprises a nucleic acid molecule encoding a GATA transcription factor.

In an embodiment of the invention, the agent that can modulate theexpression levels of a GATA transcription factor gene in a plant cellcomprises:

-   -   (a) a nucleotide sequence of SEQ ID NO:1 or a fragment or domain        thereof;    -   (b) a nucleotide sequence encoding a polypeptide of SEQ ID NO:2,        a fragment or domain thereof;    -   (c) a nucleotide sequence having substantial similarity to (a)        or (b);    -   (d) a nucleotide sequence capable of hybridizing to (a), (b) or        (c);    -   (e) a nucleotide sequence complementary to (a), (b), (c) or (d);        or    -   (f) a nucleotide sequence that is the reverse complement of (a),        (b), (c) or (d).

In a further embodiment of the invention, the nucleic acid moleculecomprises the sequence of the AT5g56860 gene of SEQ ID NO:1 or afunctional fragment thereof. In a still further embodiment of theinvention, the nucleic acid molecule comprises a sequence thathybridizes under medium stringency conditions to the AT5g56860 gene ofSEQ ID NO:1 or a functional fragment thereof. In another embodiment ofthe present invention, the nucleic acid molecule is derived from thenucleotide sequence of the At5g56860 gene of SEQ ID NO:1 and has anucleotide sequence comprising codons specific for expression in plants.In yet another embodiment of the invention, the nucleic acid molecule isthe rice orthologue of the AT5g56860 gene comprising the sequence of SEQID NO:3.

In a further embodiment of the invention, the agent that can modulatethe expression levels of a GATA transcription factor gene in a plantcell comprises:

-   -   (a) a polypeptide sequence listed in SEQ ID NO:2, or a        functional fragment, domain, repeat, or chimera thereof;    -   (b) a polypeptide sequence having substantial similarity to (a);    -   (c) a polypeptide sequence encoded by a nucleotide sequence        identical to or having substantial similarity to a nucleotide        sequence listed in SEQ ID NO:1, or a functional fragment or        domain thereof, or a sequence complementary thereto; or    -   (d) a polypeptide sequence encoded by a nucleotide sequence        capable of hybridizing under medium stringency conditions to a        nucleotide sequence listed in SEQ ID NO:1, or to a sequence        complementary thereto.

In an embodiment of the present invention, when the agent is a nucleicacid sequence, the nucleic acid sequence is expressed in a specificlocation or tissue of the plant. The location or tissue is for example,but not limited to, epidermis, root, vascular tissue, meristem, cambium,cortex, pith, leaf and/or flower. In an alternative embodiment, thelocation or tissue is a seed.

Embodiments of the present invention also relate to use of a shufflednucleic acid molecule for modulating a characteristic in a plant cell,said shuffled nucleic acid molecule containing a plurality of nucleotidesequence fragments, wherein at least one of the fragments encodes a GATAtranscription factor and wherein at least two of the plurality ofsequence fragments are in an order, from 5′ to 3′ which is not an orderin which the plurality of fragments naturally occur in a nucleic acid.In a specific embodiment, all of the fragments in a shuffled nucleicacid molecule containing a plurality of nucleotide sequence fragmentsare from a single gene. In a more specific embodiment, the plurality offragments originate from at least two different genes. In a morespecific embodiment, the shuffled nucleic acid is operably linked to apromoter sequence. Another more specific embodiment is a use of achimeric polynucleotide for modulating a characteristic in a plant cell,said chimeric polynucleotide including a promoter sequence operablylinked to the shuffled nucleic acid. In a more specific embodiment, theshuffled nucleic acid is contained within a host cell. In a furtherspecific embodiment of the invention the fragment encoding a GATAtranscription factor consists of or comprises:

(a) a nucleotide sequence of SEQ ID NO:1 or a fragment or domainthereof;

(b) a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, afragment or domain thereof;

(c) a nucleotide sequence having substantial similarity to (a) or (b);

(d) a nucleotide sequence capable of hybridizing to (a), (b) or (c);

(e) a nucleotide sequence complementary to (a), (b), (c) or (d); or

(f) a nucleotide sequence that is the reverse complement of (a), (b),(c) or (d).

Embodiments of the present invention also contemplate a use of anexpression cassette for modulating a characteristic in a plant cellincluding a promoter sequence operably linked to an isolated nucleicacid encoding a GATA transcription factor. In embodiments of theinvention the isolated nucleic acid encoding a GATA transcription factorconsists of or comprises:

(a) a nucleotide sequence of SEQ ID NO:1 or a fragment or domainthereof;

(b) a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, afragment or domain thereof;

(c) a nucleotide sequence having substantial similarity to (a) or (b);

(d) a nucleotide sequence capable of hybridizing to (a), (b) or (c)

(e) a nucleotide sequence complementary to (a), (b), (c) or (d); or

(f) a nucleotide sequence that is the reverse complement of (a), (b),(c) or (d).

Further encompassed within the invention is use of a recombinant vectorfor modulating a characteristic in a plant cell comprising an expressioncassette including a promoter sequence operably linked to an isolatednucleic acid encoding a GATA transcription factor. In embodiments of theinvention the isolated nucleic acid encoding a GATA transcription factorconsists of or comprises:

(a) a nucleotide sequence of SEQ ID NO:1 or a fragment or domainthereof;

(b) a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, afragment or domain thereof;

(c) a nucleotide sequence having substantial similarity to (a) or (b);

(d) a nucleotide sequence capable of hybridizing to (a), (b) or (c)

(e) a nucleotide sequence complementary to (a), (b), (c) or (d); or

(f) a nucleotide sequence that is the reverse complement of (a), (b),(c) or (d).

Also encompassed are uses of plant cells, which contain expressioncassettes, according to the present disclosure, and uses of plants,containing these plant cells.

In one embodiment, the expression cassette is expressed throughout theplant. In another embodiment the expression cassette is expressed in aspecific location or tissue of a plant. In a specific embodiment, thelocation or tissue may be, for example, epidermis, root, vasculartissue, meristem, cambium, cortex, pith, leaf, and flower. In analternative specific embodiment, the location or tissue is a seed.

Embodiments of the present invention also provide the use of seed andisolated product from plants for modulating a characteristic in a plantcell, which contain an expression cassette including a promoter sequenceoperably linked to an isolated nucleic acid encoding a GATAtranscription factor gene according to the present invention.

In a specific embodiment, the expression vector includes one or moreelements such as, for example, but not limited to, a promoter-enhancersequence, a selection marker sequence, an origin of replication, anepitope-tag encoding sequence, or an affinity purification-tag encodingsequence. In a more specific embodiment, the promoter-enhancer sequencemay be, for example, the CaMV 35S promoter, the CaMV 19S promoter, thetobacco PR-1a promoter, ubiquitin and the phaseolin promoter. In anotherembodiment, the promoter is operable in plants, and more specifically, aconstitutive or inducible promoter. In another specific embodiment, theselection marker sequence encodes an antibiotic resistance gene. Inanother specific embodiment, the epitope-tag sequence encodes V5, thepeptide Phe-His-His-Thr-Thr, hemagglutinin, orglutathione-S-transferase. In another specific embodiment the affinitypurification-tag sequence encodes a polyamino acid sequence or apolypeptide. In a more specific embodiment, the polyamino acid sequenceis polyhistidine. In a more specific embodiment, the polypeptide ischitin binding domain or glutathione-S-transferase. In a more specificembodiment, the affinity purification-tag sequence comprises an inteinencoding sequence.

In a specific embodiment, the expression vector is a eukaryoticexpression vector or a prokaryotic expression vector. In a more specificembodiment, the eukaryotic expression vector includes a tissue-specificpromoter. More specifically, the expression vector is operable inplants.

Embodiments of the present invention also relate to a plant modified bya method that includes introducing into a plant a nucleic acid where thenucleic acid is expressible in the plant in an amount effective toeffect the modification. The modification can be an increase or decreasein the one or more traits of interest. The modification may includeoverexpression, underexpression, antisense modulation, sensesuppression, inducible expression, inducible repression, or induciblemodulation of a gene. In an embodiment of the invention the modificationinvolved an increase or improvement in the trait of interest, forexample, nitrogen utilization.

Embodiments of the present invention provide nucleotide and amino acidsequences isolated from Arabidopsis thaliana. Particularly, the presentinvention relates to a nitrogen-regulated GATA transcription factor generequired for sugar sensing.

Embodiments of the present invention relate to an isolated nucleic acidcomprising or consisting of a nucleotide sequence comprising:

-   -   (a) a nucleotide sequence listed in SEQ ID NO:1, or a fragment        or domain, thereof;    -   (b) a nucleotide sequence having substantial similarity to (a);    -   (c) a nucleotide sequence capable of hybridizing to (a);    -   (d) a nucleotide sequence complementary to (a), (b) or (c); or    -   (e) a nucleotide sequence which is the reverse complement of        (a), (b) or (c).

In a specific embodiment, the substantial similarity is at least about65% identity, specifically about 80% identity, specifically 90%, andmore specifically at least about 95% sequence identity to the nucleotidesequence listed as SEQ ID NO:1, a fragment or domain thereof.

In a one embodiment the sequence having substantial similarity to thenucleotide sequence of SEQ ID NO:1, a fragment or domain thereof, isfrom a plant. In a specific embodiment, the plant is a dicot. In a morespecific embodiment, the dicot is selected from the group consisting ofsoybean, tobacco or cotton. In another specific embodiment, the plant isa gymnosperm. In another specific embodiment, the plant is a monocot. Ina more specific embodiment, the monocot is a cereal. In a more specificembodiment, the cereal may be, for example, maize, wheat, barley, oats,rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff,milo, flax, gramma grass, Tripsacum sp., or teosinte.

In one embodiment the nucleic acid is expressed in a specific locationor tissue of a plant. The location or tissue is for example, but notlimited to, epidermis, root, vascular tissue, meristem, cambium, cortex,pith, leaf, and flower. In an alternative embodiment, the location ortissue is a seed. In another embodiment, the nucleic acid encodes apolypeptide involved in a function such as, for example, but not limitedto, carbon, nitrogen and/or sulfur metabolism, nitrogen utilization,nitrogen assimilation, photosynthesis, signal transduction, cell growth,reproduction, disease resistance, abiotic stress tolerance, nutritionalcomposition, gene regulation, and/or differentiation.

In a specific embodiment, the isolated nucleic acid comprises orconsists of a nucleotide sequence capable of hybridizing to a nucleotidesequence listed in SEQ ID NO:1 or a fragment or domain thereof. In aspecific embodiment, hybridization allows the sequence to form a duplexat medium or high stringency. Embodiments of the present invention alsoencompass a nucleotide sequence complementary to a nucleotide sequenceof SEQ ID NO:1 or a fragment or domain thereof. Embodiments of thepresent invention further encompass a nucleotide sequence complementaryto a nucleotide sequence that has substantial similarity or is capableof hybridizing to a nucleotide sequence of SEQ ID NO:1 or a fragment ordomain thereof.

In a specific embodiment, the nucleotide sequence having substantialsimilarity is an allelic variant of the nucleotide sequence of SEQ IDNO:1a fragment or domain thereof. In an alternate embodiment, thesequence having substantial similarity is a naturally occurring variant.In another alternate embodiment, the sequence having substantialsimilarity is a polymorphic variant of the nucleotide sequence of SEQ IDNO:1 or a fragment or domain thereof.

In a specific embodiment, the isolated nucleic acid contains a pluralityof regions having the nucleotide sequence of SEQ ID NO:1 or exon ordomain thereof.

In a specific embodiment, the isolated nucleic acid contains apolypeptide-encoding sequence. In a more specific embodiment, thepolypeptide-encoding sequence contains a 20 base pair nucleotide portionidentical in sequence to a consecutive 20 base pair nucleotide portionof a nucleic acid sequence of SEQ ID NO:1. In a more specificembodiment, the polypeptide contains a polypeptide sequence of SEQ IDNO:2, or a fragment thereof. In a more specific embodiment, thepolypeptide is a plant polypeptide. In a more specific embodiment, theplant is a dicot. In a more specific embodiment, the plant is agymnosperm. In a more specific embodiment, the plant is a monocot. In amore specific embodiment, the monocot is a cereal. In a more specificembodiment, the cereal may be, for example, maize, wheat, barley, oats,rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff,miloflax, gramma grass, Tripsacum, and teosinte.

In one embodiment, the polypeptide is expressed throughout the plant. Ina more specific embodiment, the polypeptide is expressed in a specificlocation or tissue of a plant. In a more specific embodiment, thelocation or tissue may be, for example, epidermis, root, vasculartissue, meristem, cambium, cortex, pith, leaf, and flower. In a mostspecific embodiment, the location or tissue is a seed.

In a specific embodiment, the sequence of the isolated nucleic acidencodes a polypeptide useful for generating an antibody havingimmunoreactivity against a polypeptide encoded by a nucleotide sequenceof SEQ ID NO:2, or fragment or domain thereof.

In a specific embodiment, the sequence having substantial similaritycontains a deletion or insertion of at least one nucleotide. In a morespecific embodiment, the deletion or insertion is of less than aboutthirty nucleotides. In a most specific embodiment, the deletion orinsertion is of less than about five nucleotides.

In a specific embodiment, the sequence of the isolated nucleic acidhaving substantial similarity comprises or consists of a substitution inat least one codon. In a specific embodiment, the substitution isconservative.

Embodiments of the present invention also relate to an isolated nucleicacid molecule comprising or consisting of a nucleotide sequence, itscomplement, or its reverse complement, encoding a polypeptide including:

-   -   (a) a polypeptide sequence of SEQ ID NO:2, or a fragment,        domain, repeat, or chimera thereof;    -   (b) a polypeptide sequence having substantial similarity to (a);    -   (c) a polypeptide sequence encoded by a nucleotide sequence        identical to or having substantial similarity to a nucleotide        sequence of SEQ ID NO:1, or a fragment or domain thereof, or a        sequence complementary thereto;    -   (d) a polypeptide sequence encoded by a nucleotide sequence        capable of hybridizing under medium stringency conditions to a        nucleotide sequence of SEQ ID NO:1 or a sequence complementary        thereto; or    -   (e) a functional fragment of (a), (b), (c) or (d).

In another specific embodiment, the polypeptide having substantialsimilarity is an allelic variant of a polypeptide sequence of SEQ IDNO:2, or a fragment, domain, repeat or chimera thereof. In anotherspecific embodiment, the isolated nucleic acid includes a plurality ofregions from the polypeptide sequence encoded by a nucleotide sequenceidentical to or having substantial similarity to a nucleotide sequenceof SEQ ID NO:1, or fragment or domain thereof, or a sequencecomplementary thereto.

In another specific embodiment, the polypeptide is a polypeptidesequence of SEQ ID NO:2. In another specific embodiment, the polypeptideis a functional fragment or domain. In yet another specific embodiment,the polypeptide is a chimera, where the chimera may include functionalprotein domains, including domains, repeats, post-translationalmodification sites, or other features. In a more specific embodiment,the polypeptide is a plant polypeptide. In a more specific embodiment,the plant is a dicot. In a more specific embodiment, the plant is agymnosperm. In a more specific embodiment, the plant is a monocot. In amore specific embodiment, the monocot is a cereal. In a more specificembodiment, the cereal may be, for example, maize, wheat, barley, oats,rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff,milo, flax, gramma grass, Tripsacum, and teosinte.

In a specific embodiment, the polypeptide is expressed in a specificlocation or tissue of a plant. In a more specific embodiment, thelocation or tissue may be, for example, epidermis, root, vasculartissue, meristem, cambium, cortex, pith, leaf, and flower. In anotherspecific embodiment, the location or tissue is a seed.

In a specific embodiment, the polypeptide sequence encoded by anucleotide sequence having substantial similarity to a nucleotidesequence of SEQ ID NO:1 or a fragment or domain thereof or a sequencecomplementary thereto, includes a deletion or insertion of at least onenucleotide. In a more specific embodiment, the deletion or insertion isof less than about thirty nucleotides. In a most specific embodiment,the deletion or insertion is of less than about five nucleotides.

In a specific embodiment, the polypeptide sequence encoded by anucleotide sequence having substantial similarity to a nucleotidesequence of SEQ ID NO:1, or a fragment or domain thereof or a sequencecomplementary thereto, includes a substitution of at least one codon. Ina more specific embodiment, the substitution is conservative.

In a specific embodiment, the polypeptide sequences having substantialsimilarity to the polypeptide sequence of SEQ ID NO:2 or a fragment,domain, repeat, or chimeras thereof includes a deletion or insertion ofat least one amino acid.

In a specific embodiment, the polypeptide sequences having substantialsimilarity to the polypeptide sequence of SEQ ID NO:2 or a fragment,domain, repeat, or chimeras thereof includes a substitution of at leastone amino acid.

Embodiments of the present invention also relate to a shuffled nucleicacid containing a plurality of nucleotide sequence fragments, wherein atleast one of the fragments corresponds to a region of a nucleotidesequence of SEQ ID NO:1 and wherein at least two of the plurality ofsequence fragments are in an order, from 5′ to 3′ which is not an orderin which the plurality of fragments naturally occur in a nucleic acid.In a more specific embodiment, all of the fragments in a shufflednucleic acid containing a plurality of nucleotide sequence fragments arefrom a single gene. In a more specific embodiment, the plurality offragments originates from at least two different genes. In a morespecific embodiment, the shuffled nucleic acid is operably linked to apromoter sequence. Another more specific embodiment is a chimericpolynucleotide including a promoter sequence operably linked to theshuffled nucleic acid. In a more specific embodiment, the shufflednucleic acid is contained within a host cell.

Embodiments of the present invention also contemplate an expressioncassette including a promoter sequence operably linked to an isolatednucleic acid containing a nucleotide sequence including:

-   -   a) a nucleotide sequence of SEQ ID NO:1 or a fragment or domain        thereof;    -   (b) a nucleotide sequence encoding a polypeptide of SEQ ID NO:2,        a fragment or domain thereof;    -   (c) a nucleotide sequence having substantial similarity to (a)        or (b);    -   (d) a nucleotide sequence capable of hybridizing to (a), (b) or        (c);    -   (e) a nucleotide sequence complementary to (a), (b), (c) or (d);        or    -   (f) a nucleotide sequence that is the reverse complement of (a),        (b), (c) or (d).

Further encompassed within the invention is a recombinant vectorcomprising an expression cassette according to embodiments of thepresent invention. Also encompassed are plant cells, which containexpression cassettes, according to the present disclosure, and plants,containing these plant cells. In a specific embodiment, the plant is adicot. In a more specific embodiment, the dicot is selected from thegroup consisting of soybean, tobacco or cotton. In another specificembodiment, the plant is a gymnosperm. In another specific embodiment,the plant is a monocot. In a more specific embodiment, the monocot is acereal. In a more specific embodiment, the cereal may be, for example,maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale,einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum andteosinte.

In one embodiment, the expression cassette is expressed throughout theplant. In another embodiment, the expression cassette is expressed in aspecific location or tissue of a plant. In a specific embodiment, thelocation or tissue may be, for example, epidermis, root, vasculartissue, meristem, cambium, cortex, pith, leaf, and flower. In analternative specific embodiment, the location or tissue is a seed.

In one embodiment, the expression cassette is involved in a functionsuch as, for example, but not limited to, carbon, nitrogen and/or sulfurmetabolism, nitrogen utilization, nitrogen assimilation, photosynthesis,signal transduction, cell growth, reproduction, disease resistance,abiotic stress tolerance, nutritional composition, gene regulation,and/or differentiation. In a more specific embodiment, the chimericpolypeptide is involved in a function such as, nitrogen utilization,abiotic stress tolerance, enhanced yield, disease resistance and/ornutritional composition.

In one embodiment, the plant contains a modification to a phenotype ormeasurable characteristic of the plant, the modification beingattributable to the expression of at least one gene contained in theexpression cassette. In a specific embodiment, the modification may be,for example, carbon, nitrogen and/or sulfur metabolism, nitrogenutilization, nitrogen assimilation, photosynthesis, signal transduction,cell growth, reproduction, disease resistance, abiotic stress tolerance,nutritional composition, gene regulation, and/or differentiation.

Embodiments of the present invention also provide seed and isolatedproduct from plants which contain an expression cassette including apromoter sequence operably linked to an isolated nucleic acid containinga nucleotide sequence including:

(a) a nucleotide sequence of SEQ ID NO:1 or a fragment or domainthereof;

(b) a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, afragment or domain thereof;

(c) a nucleotide sequence having substantial similarity to (a) or (b);

(d) a nucleotide sequence capable of hybridizing to (a), (b) or (c);

(e) a nucleotide sequence complementary to (a), (b), (c) or (d); or

(f) a nucleotide sequence that is the reverse complement of (a), (b),(c) or (d) according to the present disclosure.

In a specific embodiment the isolated product includes an enzyme, anutritional protein, a structural protein, an amino acid, a lipid, afatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid, acarotenoid, a propanoid, a steroid, a pigment, a vitamin and a planthormone.

Embodiments of the present invention also relate to isolated productsproduced by expression of an isolated nucleic acid containing anucleotide sequence including:

-   -   (a) a nucleotide sequence of SEQ ID NO:1, or fragment or domain        thereof;    -   (b) a nucleotide sequence encoding a polypeptide of SEQ ID NO:2,        or a fragment or domain thereof;    -   (c) a nucleotide sequence having substantial similarity to (a)        or (b);    -   (d) a nucleotide sequence capable of hybridizing to (a) or (b);    -   (e) a nucleotide sequence complementary to (a), (b), (c) or (d);        or    -   (f) a nucleotide sequence that is the reverse complement of        (a), (b) (c) or (d) according to the present disclosure.

In a specific embodiment, the product is produced in a plant. In anotherspecific embodiment, the product is produced in cell culture. In anotherspecific embodiment, the product is produced in a cell-free system. Inanother specific embodiment, the product includes an enzyme, anutritional protein, a structural protein, an amino acid, a lipid, afatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid, acarotenoid, a propanoid, a steroid, a pigment, a vitamin and a planthormone.

In a specific embodiment, the product is a polypeptide containing anamino acid sequence of SEQ ID NO:2. In a more specific embodiment, theprotein is an transcription factor.

Embodiments of the present invention further relate to an isolatedpolynucleotide including a nucleotide sequence of at least 10 bases,which sequence is identical, complementary, or substantially similar toa region of any sequence of SEQ ID NO:1, and wherein the polynucleotideis adapted for any of numerous uses.

In a specific embodiment, the polynucleotide is used as a chromosomalmarker. In another specific embodiment, the polynucleotide is used as amarker for RFLP analysis. In another specific embodiment, thepolynucleotide is used as a marker for quantitative trait linkedbreeding. In another specific embodiment, the polynucleotide is used asa marker for marker-assisted breeding. In another specific embodiment,the polynucleotide is used as a bait sequence in a two-hybrid system toidentify sequence-encoding polypeptides interacting with the polypeptideencoded by the bait sequence. In another specific embodiment, thepolynucleotide is used as a diagnostic indicator for genotyping oridentifying an individual or population of individuals. In anotherspecific embodiment, the polynucleotide is used for genetic analysis toidentify boundaries of genes or exons.

Embodiments of the present invention also relate to an expression vectorcomprising or consisting of a nucleic acid molecule including:

-   -   (a) a nucleic acid encoding a polypeptide as listed in SEQ ID        NO:2    -   (b) a fragment, one or more domains, or featured regions of SEQ        ID NO:1; or    -   (c) a complete nucleic acid sequence listed in SEQ ID NO:1, or a        fragment thereof, in combination with a heterologous sequence.

In a specific embodiment, the expression vector includes one or moreelements such as, for example, but not limited to, a promoter-enhancersequence, a selection marker sequence, an origin of replication, anepitope-tag encoding sequence, or an affinity purification-tag encodingsequence. In a more specific embodiment, the promoter-enhancer sequencemay be, for example, the CaMV 35S promoter, the CaMV 19S promoter, thetobacco PR-1a promoter, ubiquitin and the phaseolin promoter. In anotherembodiment, the promoter is operable in plants, and more specifically, aconstitutive or inducible promoter. In another specific embodiment, theselection marker sequence encodes an antibiotic resistance gene. Inanother specific embodiment, the epitope-tag sequence encodes V5, thepeptide Phe-His-His-Thr-Thr, hemagglutinin, orglutathione-S-transferase. In another specific embodiment the affinitypurification-tag sequence encodes a polyamino acid sequence or apolypeptide. In a more specific embodiment, the polyamino acid sequenceis polyhistidine. In a more specific embodiment, the polypeptide ischitin binding domain or glutathione-S-transferase. In a more specificembodiment, the affinity purification-tag sequence comprises an inteinencoding sequence.

In a specific embodiment, the expression vector is a eukaryoticexpression vector or a prokaryotic expression vector. In a more specificembodiment, the eukaryotic expression vector includes a tissue-specificpromoter. More specifically, the expression vector is operable inplants.

Embodiments of the present invention also relate to a cell comprising orconsisting of a nucleic acid construct comprising an expression vectorand a nucleic acid including a nucleic acid encoding a polypeptide aslisted in SEQ ID NO:2, or a nucleic acid sequence listed in SEQ ID NO:1,or a segment thereof, in combination with a heterologous sequence.

In a specific embodiment, the cell is a bacterial cell, a fungal cell, aplant cell, or an animal cell. In a specific embodiment, the cell is aplant cell. In a more specific embodiment, the polypeptide is expressedin a specific location or tissue of a plant. In a most specificembodiment, the location or tissue may be, for example, epidermis, root,vascular tissue, meristem, cambium, cortex, pith, leaf, and flower. Inan alternate most specific embodiment, the location or tissue is a seed.In a specific embodiment, the polypeptide is involved in a function suchas, for example, carbon, nitrogen and/or sulfur metabolism, nitrogenutilization, nitrogen assimilation, photosynthesis, signal transduction,cell growth, reproduction, disease resistance, abiotic stress tolerance,nutritional composition, gene regulation, and/or differentiation.

Embodiments of the present invention also relate to polypeptides encodedby the isolated nucleic acid molecules of the present disclosureincluding a polypeptide containing a polypeptide sequence encoded by anisolated nucleic acid containing a nucleotide sequence including:

-   -   (a) a nucleotide sequence listed in SEQ ID NO:1 or an exon or        domain thereof;    -   (b) a nucleotide sequence having substantial similarity to (a);    -   (c) a nucleotide sequence capable of hybridizing to (a);    -   (d) a nucleotide sequence complementary to (a), (b) or (c); or    -   (e) a nucleotide sequence which is the reverse complement of        (a), (b) or (c);    -   (f) or a functional fragment thereof.

A polypeptide containing a polypeptide sequence encoded by an isolatednucleic acid containing a nucleotide sequence, its complement, or itsreverse complement, encoding a polypeptide including a polypeptidesequence including:

-   -   (a) a polypeptide sequence listed in SEQ ID NO:2, or a domain,        repeat, or chimeras thereof;    -   (b) a polypeptide sequence having substantial similarity to (a);    -   (c) a polypeptide sequence encoded by a nucleotide sequence        identical to or having substantial similarity to a nucleotide        sequence listed SEQ ID NO:1, or an exon or domain thereof, or a        sequence complementary thereto;    -   (d) a polypeptide sequence encoded by a nucleotide sequence        capable of hybridizing under medium stringency conditions to a        nucleotide sequence listed in SEQ ID NO:1 or to a sequence        complementary thereto; or    -   (e) a functional fragment of (a), (b), (c) or (d);    -   (f) or a functional fragment thereof.

Embodiments of the present invention contemplate a polypeptidecontaining a polypeptide sequence encoded by an isolated nucleic acidwhich includes a shuffled nucleic acid containing a plurality ofnucleotide sequence fragments, wherein at least one of the fragmentscorresponds to a region of a nucleotide sequence listed SEQ ID NO:1, andwherein at least two of the plurality of sequence fragments are in anorder, from 5′ to 3′ which is not an order in which the plurality offragments naturally occur in a nucleic acid, or functional fragmentthereof.

Embodiments of the present invention contemplate a polypeptidecontaining a polypeptide sequence encoded by an isolated polynucleotidecontaining a nucleotide sequence of at least 10 bases, which sequence isidentical, complementary, or substantially similar to a region of any ofsequences of SEQ ID NO:1, or functional fragment thereof and wherein thepolynucleotide is adapted for a use including:

-   -   (a) use as a chromosomal marker to identify the location of the        corresponding or complementary polynucleotide on a native or        artificial chromosome;    -   (b) use as a marker for RFLP analysis;    -   (c) use as a marker for quantitative trait linked breeding;    -   (d) use as a marker for marker-assisted breeding;    -   (e) use as a bait sequence in a two-hybrid system to identify        sequence encoding polypeptides interacting with the polypeptide        encoded by the bait sequence;    -   (f) use as a diagnostic indicator for genotyping or identifying        an individual or population of individuals; or    -   (g) use for genetic analysis to identify boundaries of genes or        exons.

Embodiments of the present invention also contemplate an isolatedpolypeptide containing a polypeptide sequence including:

-   -   (a) a polypeptide sequence listed SEQ ID NO:2, or exon or domain        thereof;    -   (b) a polypeptide sequence having substantial similarity to (a);    -   (c) a polypeptide sequence encoded by a nucleotide sequence        identical to or having substantial similarity to a nucleotide        sequence SEQ ID NO:1, or an exon or domain thereof or a sequence        complementary thereto;    -   (d) a polypeptide sequence encoded by a nucleotide sequence        capable of hybridizing under medium stringency conditions to a        nucleotide sequence listed in SEQ ID NO:1, or to a sequence        complementary thereto; or    -   (e) a functional fragment of (a), (b), (c) or (d).

In a specific embodiment, the substantial similarity is at least about65% identity. In a more specific embodiment, the substantial similarityis at least about 80% identity. In a most specific embodiment, thesubstantial similarity is at least about 95% identity. In a specificembodiment, the substantial similarity is at least three percent greaterthan the percent identity to the closest homologous sequence listed inany of the Sequence Listings.

In a specific embodiment, the sequence having substantial similarity isfrom a plant. In a more specific embodiment, the plant is a dicot. In amore specific embodiment, the plant is a gymnosperm. In a more specificembodiment, the plant is a monocot. In a more specific embodiment, themonocot is a cereal. In a more specific embodiment, the cereal may be,for example, maize, wheat, barley, oats, rye, millet, sorghum,triticale, secale, einkorn, spelt, emmer, teff, milo, flax, grammagrass, Tripsacum and teosinte.

In a specific embodiment, the polypeptide is expressed in a specificlocation or tissue of a plant. In a more specific embodiment, thelocation or tissue may be, for example, epidermis, root, vasculartissue, meristem, cambium, cortex, pith, leaf, and flower. In anotherspecific embodiment, the location or tissue is a seed. In a specificembodiment, the polypeptide is involved in a function such as, forexample, carbon, nitrogen and/or sulfur metabolism, nitrogenutilization, nitrogen assimilation, photosynthesis, signal transduction,cell growth, reproduction, disease resistance, abiotic stress tolerance,nutritional composition, gene regulation, and/or differentiation.

In a specific embodiment, hybridization of a polypeptide sequenceencoded by a nucleotide sequence identical to or having substantialsimilarity to a nucleotide sequence listed in SEQ ID NO:1, or an exon ordomain thereof, or a sequence complementary thereto, or a polypeptidesequence encoded by a nucleotide sequence capable of hybridizing undermedium stringency conditions to a nucleotide sequence listed SEQ IDNO:1, or to a sequence complementary thereto, allows the sequence toform a duplex at medium or high stringency.

In a specific embodiment, a polypeptide having substantial similarity toa polypeptide sequence listed in SEQ ID NO:2, or exon or domain thereof,is an allelic variant of the polypeptide sequence listed in SEQ ID NO:2.In another specific embodiment, a polypeptide having substantialsimilarity to a polypeptide sequence listed in SEQ ID NO:2, or exon ordomain thereof is a naturally occurring variant of the polypeptidesequence listed in SEQ ID NO:2. In another specific embodiment, apolypeptide having substantial similarity to a polypeptide sequencelisted in SEQ ID NO:2, or exon or domain thereof is a polymorphicvariant of the polypeptide sequence listed in SEQ ID NO:2.

In an alternate specific embodiment, the sequence having substantialsimilarity contains a deletion or insertion of at least one amino acid.In a more specific embodiment, the deletion or insertion is of less thanabout ten amino acids. In a most specific embodiment, the deletion orinsertion is of less than about three amino acids.

In a specific embodiment, the sequence having substantial similarityencodes a substitution in at least one amino acid.

Also contemplated is a method of producing a plant comprising amodification thereto, including the steps of: (1) providing a nucleicacid which is an isolated nucleic acid containing a nucleotide sequenceincluding:

-   -   (a) a nucleotide sequence listed SEQ ID NO:1, or exon or domain        thereof;    -   (b) a nucleotide sequence having substantial similarity to (a);    -   (c) a nucleotide sequence capable of hybridizing to (a);    -   (d) a nucleotide sequence complementary to (a), (b) or (c); or    -   (e) a nucleotide sequence which is the reverse complement of        (a), (b) or (c);        and (2) introducing the nucleic acid into the plant, wherein the        nucleic acid is expressible in the plant in an amount effective        to effect the modification. In one embodiment, the modification        comprises an altered characteristic in the plant, wherein the        characteristic corresponds to the nucleic acid introduced into        the plant. In other specific embodiments the characteristic        corresponds to carbon, nitrogen and/or sulfur metabolism,        nitrogen utilization, nitrogen assimilation, photosynthesis,        signal transduction, cell growth, reproduction, disease        resistance, abiotic stress tolerance, nutritional composition,        gene regulation, and/or differentiation.

In another embodiment, the modification includes an increased ordecreased expression or accumulation of a product of the plant.Specifically, the product is a natural product of the plant. Equallyspecifically, the product is a new or altered product of the plant.Specifically, the product comprises a GATA transcription factor.

Also encompassed within the presently disclosed invention is a method ofproducing a recombinant protein, comprising the steps of:

(a) growing recombinant cells comprising a nucleic acid construct undersuitable growth conditions, the construct comprising an expressionvector and a nucleic acid including: a nucleic acid encoding a proteinas listed in SEQ ID NO:2, or a nucleic acid sequence listed in SEQ IDNO:1, or segments thereof; and

(b) isolating from the recombinant cells the recombinant proteinexpressed thereby.

Embodiments of the present invention provide a method of producing arecombinant protein in which the expression vector includes one or moreelements including a promoter-enhancer sequence, a selection markersequence, an origin of replication, an epitope-tag encoding sequence,and an affinity purification-tag encoding sequence. In one specificembodiment, the nucleic acid construct includes an epitope-tag encodingsequence and the isolating step includes use of an antibody specific forthe epitope-tag. In another specific embodiment, the nucleic acidconstruct contains a polyamino acid encoding sequence and the isolatingstep includes use of a resin comprising a polyamino acid bindingsubstance, specifically where the polyamino acid is polyhistidine andthe polyamino binding resin is nickel-charged agarose resin in yetanother specific embodiment, the nucleic acid construct contains apolypeptide encoding sequence and the isolating step includes the use ofa resin containing a polypeptide binding substance, specifically wherethe polypeptide is a chitin binding domain and the resin containschitin-sepharose.

Embodiments of the present invention also relate to a plant modified bya method that includes introducing into a plant a nucleic acid where thenucleic acid is expressible in the plant in an amount effective toeffect the modification. The modification can be, for example, carbon,nitrogen and/or sulfur metabolism, nitrogen utilization, nitrogenassimilation, photosynthesis, signal transduction, cell growth,reproduction, disease resistance, abiotic stress tolerance, nutritionalcomposition, gene regulation, and/or differentiation. In one embodiment,the modified plant has increased or decreased resistance to anherbicide, a stress, or a pathogen. In another embodiment, the modifiedplant has enhanced or diminished requirement for light, water, nitrogen,or trace elements. In yet another embodiment, the modified plant isenriched for an essential amino acid as a proportion of a proteinfraction of the plant. The protein fraction may be, for example, totalseed protein, soluble protein, insoluble protein, water-extractableprotein, and lipid-associated protein. The modification may includeoverexpression, underexpression, antisense modulation, sensesuppression, inducible expression, inducible repression, or induciblemodulation of a gene.

The invention further relates to a seed from a modified plant or anisolated product of a modified plant, where the product may be anenzyme, a nutritional protein, a structural protein, an amino acid, alipid, a fatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid,a carotenoid, a propanoid, a steroid, a pigment, a vitamin and a planthormone.

The above Summary of Invention lists several embodiments of theinvention, and in many cases lists variations and permutations of theseembodiments. The Summary is merely exemplary of the numerous and variedembodiments Mention of one or more specific features of a givenembodiment is likewise exemplary. Such embodiment can typically existwith or without the feature(s) mentioned; likewise, those features canbe applied to other embodiments of the invention, whether listed in thisSummary or not. To avoid excessive repetition, this Summary does notlist or suggest all possible combinations of such features.

For purposes of summarizing the invention and the advantages achievedover the prior art, certain objects and advantages of the invention havebeen described above. Of course, it is to be understood that notnecessarily all such objects or advantages may be achieved in accordancewith any particular embodiment of the invention. Thus, for example,those skilled in the art will recognize that the invention may beembodied or carried out in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objects or advantages as may be taught or suggestedherein.

Further aspects, features and advantages of this invention will becomeapparent from the detailed description of the specific embodiments thatfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 and SEQ ID NO:1 shows the CDS sequence of full length At5g56860.

FIG. 2 and SEQ ID NO:2 shows the amino acid sequence of At5g56860.

FIG. 3 and SEQ ID NO:3 shows the nucleic acid sequence of the riceorthologue of At5g56860.

FIG. 4 is a construct of the plasmid containing CaMV 35S promoter, GNCcDNA and the nopaline synthase terminator (nos) between the right (RB)and left (LB) borders of the T-DNA.

DEFINITIONS

For clarity, certain terms used in the specification are defined andpresented as follows:

“Associated with/operatively linked” refer to two nucleic acid sequencesthat are related physically or functionally. For example, a promoter orregulatory DNA sequence is said to be “associated with” a DNA sequencethat codes for an RNA or a protein if the two sequences are operativelylinked, or situated such that the regulator DNA sequence will affect theexpression level of the coding or structural DNA sequence.

A “chimeric construct” is a recombinant nucleic acid sequence in which apromoter or regulatory nucleic acid sequence is operatively linked to,or associated with, a nucleic acid sequence that codes for an mRNA orwhich is expressed as a protein, such that the regulatory nucleic acidsequence is able to regulate transcription or expression of theassociated nucleic acid sequence. The regulatory nucleic acid sequenceof the chimeric construct is not normally operatively linked to theassociated nucleic acid sequence as found in nature.

A “co-factor” is a natural reactant, such as an organic molecule or ametal ion, required in an enzyme-catalyzed reaction. A co-factor is e.g.NAD(P), riboflavin (including FAD and FMN), folate, molybdopterin,thiamin, biotin, lipoic acid, pantothenic acid and coenzyme A,S-adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone.Optionally, a co-factor can be regenerated and reused.

A “coding sequence” is a nucleic acid sequence that is transcribed intoRNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.Specifically the RNA is then translated in an organism to produce aprotein.

Complementary: “complementary” refers to two nucleotide sequences thatcomprise antiparallel nucleotide sequences capable of pairing with oneanother upon formation of hydrogen bonds between the complementary baseresidues in the antiparallel nucleotide sequences.

Enzyme activity: means herein the ability of an enzyme to catalyze theconversion of a substrate into a product. A substrate for the enzymecomprises the natural substrate of the enzyme but also comprisesanalogues of the natural substrate, which can also be converted, by theenzyme into a product or into an analogue of a product. The activity ofthe enzyme is measured for example by determining the amount of productin the reaction after a certain period of time, or by determining theamount of substrate remaining in the reaction mixture after a certainperiod of time. The activity of the enzyme is also measured bydetermining the amount of an unused co-factor of the reaction remainingin the reaction mixture after a certain period of time or by determiningthe amount of used co-factor in the reaction mixture after a certainperiod of time. The activity of the enzyme is also measured bydetermining the amount of a donor of free energy or energy-rich molecule(e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine)remaining in the reaction mixture after a certain period of time or bydetermining the amount of a used donor of free energy or energy-richmolecule (e.g. ADP, pyruvate, acetate or creatine) in the reactionmixture after a certain period of time.

Expression Cassette: “Expression cassette” as used herein means anucleic acid molecule capable of directing expression of a particularnucleotide sequence in an appropriate host cell, comprising a promoteroperatively linked to the nucleotide sequence of interest which isoperatively linked to termination signals. It also typically comprisessequences required for proper translation of the nucleotide sequence.The coding region usually codes for a protein of interest but may alsocode for a functional RNA of interest, for example antisense RNA or anontranslated RNA, in the sense or antisense direction. The expressioncassette comprising the nucleotide sequence of interest may be chimeric,meaning that at least one of its components is heterologous with respectto at least one of its other components. The expression cassette mayalso be one that is naturally occurring but has been obtained in arecombinant form useful for heterologous expression. Typically, however,the expression cassette is heterologous with respect to the host, i.e.,the particular DNA sequence of the expression cassette does not occurnaturally in the host cell and must have been introduced into the hostcell or an ancestor of the host cell by a transformation event. Theexpression of the nucleotide sequence in the expression cassette may beunder the control of a constitutive promoter or of an inducible promoterthat initiates transcription only when the host cell is exposed to someparticular external stimulus. In the case of a multicellular organism,such as a plant, the promoter can also be specific to a particulartissue or organ or stage of development.

The term “functional fragment” as used herein in relation to a nucleicacid or protein sequence means a fragment or portion of the sequencethat retains the function of the full length sequence.

Gene: the term “gene” is used broadly to refer to any segment of DNAassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.Genes also include nonexpressed DNA segments that, for example, formrecognition sequences for other proteins. Genes can be obtained from avariety of sources, including cloning from a source of interest orsynthesizing from known or predicted sequence information, and mayinclude sequences designed to have desired parameters.

Heterologous/exogenous: The terms “heterologous” and “exogenous” whenused herein to refer to a nucleic acid sequence (e.g. a DNA sequence) ora gene, refer to a sequence that originates from a source foreign to theparticular host cell or, if from the same source, is modified from itsoriginal form. Thus, a heterologous gene in a host cell includes a genethat is endogenous to the particular host cell but has been modifiedthrough, for example, the use of DNA shuffling. The terms also includenon-naturally occurring multiple copies of a naturally occurring DNAsequence. Thus, the terms refer to a DNA segment that is foreign orheterologous to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is not ordinarilyfound. Exogenous DNA segments are expressed to yield exogenouspolypeptides.

A “homologous” nucleic acid (e.g. DNA) sequence is a nucleic acid (e.g.DNA) sequence naturally associated with a host cell into which it isintroduced.

Hybridization: The phrase “hybridizing specifically to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s)substantially” refers to complementary hybridization between a probenucleic acid and a target nucleic acid and embraces minor mismatchesthat can be accommodated by reducing the stringency of the hybridizationmedia to achieve the desired detection of the target nucleic acidsequence.

Inhibitor: a chemical substance that inactivates the enzymatic activityof a protein such as a biosynthetic enzyme, receptor, signaltransduction protein, structural gene product, or transport protein. Theterm “herbicide” (or “herbicidal compound”) is used herein to define aninhibitor applied to a plant at any stage of development, whereby theherbicide inhibits the growth of the plant or kills the plant.

Interaction: quality or state of mutual action such that theeffectiveness or toxicity of one protein or compound on another proteinis inhibitory (antagonists) or enhancing (agonists).

A nucleic acid sequence is “isocoding with” a reference nucleic acidsequence when the nucleic acid sequence encodes a polypeptide having thesame amino acid sequence as the polypeptide encoded by the referencenucleic acid sequence.

Isogenic: plants that are genetically identical, except that they maydiffer by the presence or absence of a heterologous DNA sequence.

Isolated: in the context of the present invention, an isolated DNAmolecule or an isolated enzyme is a DNA molecule or enzyme that, byhuman intervention, exists apart from its native environment and istherefore not a product of nature. An isolated DNA molecule or enzymemay exist in a purified form or may exist in a non-native environmentsuch as, for example, in a transgenic host cell.

Mature protein: protein from which the transit peptide, signal peptide,and/or propeptide portions have been removed.

Minimal Promoter: the smallest piece of a promoter, such as a TATAelement, that can support any transcription. A minimal promotertypically has greatly reduced promoter activity in the absence ofupstream activation. In the presence of a suitable transcription factor,the minimal promoter functions to permit transcription.

Modified Enzyme Activity: enzyme activity different from that whichnaturally occurs in a plant (i.e. enzyme activity that occurs naturallyin the absence of direct or indirect manipulation of such activity byman), which is tolerant to inhibitors that inhibit the naturallyoccurring enzyme activity.

Native: refers to a gene that is present in the genome of anuntransformed plant cell.

Naturally occurring: the term “naturally occurring” is used to describean object that can be found in nature as distinct from beingartificially produced by man. For example, a protein or nucleotidesequence present in an organism (including a virus), which can beisolated from a source in nature and which has not been intentionallymodified by man in the laboratory, is naturally occurring.

Nucleic acid: the term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608(1985), Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). The terms“nucleic acid” or “nucleic acid sequence” may also be usedinterchangeably with gene, cDNA, and mRNA encoded by a gene.

“ORF” means open reading frame.

Percent identity: the phrases “percent identityl” or “percentidentical,” in the context of two nucleic acid or protein sequences,refers to two or more sequences or subsequences that have for example60%, specifically 70%, more specifically 80%, still more specifically90%, even more specifically 95%, and most specifically at least 99%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Specifically, the percentidentity exists over a region of the sequences that is at least about 50residues in length, more specifically over a region of at least about100 residues, and most specifically the percent identity exists over atleast about 150 residues. In an especially specific embodiment, thepercent identity exists over the entire length of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequencers) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48: 443 (1970), by the search for similarity method ofPearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally, Ausubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website. This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., 1990). Theseinitial neighborhood word hits act as seeds for initiating search tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when the cumulative alignment scorefalls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below cue to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses a defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA 89: 10915 (1989)).

In addition to calculating percent sequence identity; the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,more specifically less than about 0.01, and most specifically less thanabout 0.001.

Pre-protein: protein that is normally targeted to a cellular organelle,such as a chloroplast, and still comprises its native transit peptide.

Purified: the term “purified,” when applied to a nucleic acid orprotein, denotes that the nucleic acid or protein is essentially free ofother cellular components with which it is associated in the naturalstate. It is specifically in a homogeneous state although it can be ineither a dry or aqueous solution. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. The term “purified” denotes that a nucleic acidor protein gives rise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at leastabout 50% pure, more specifically at least about 85% pure, and mostspecifically at least about 99% pure.

Two nucleic acids are “recombined” when sequences from each of the twonucleic acids are combined in a progeny nucleic acid. Two sequences are“directly” recombined when both of the nucleic acids are substrates forrecombination. Two sequences are “indirectly recombined” when thesequences are recombined using an intermediate such as a cross-overoligonucleotide. For indirect recombination, no more than one of thesequences is an actual substrate for recombination, and in some cases,neither sequence is a substrate for recombination.

“Regulatory elements” refer to sequences involved in controlling theexpression of a nucleotide sequence. Regulatory elements comprise apromoter operatively linked to the nucleotide sequence of interest andtermination signals. They also typically encompass sequences requiredfor proper translation of the nucleotide sequence.

Significant Increase: an increase in enzymatic activity that is largerthan the margin of error inherent in the measurement technique,specifically an increase by about 2-fold or greater of the activity ofthe wild-type enzyme in the presence of the inhibitor, more specificallyan increase by about 5-fold or greater, and most specifically anincrease by about 10-fold or greater.

Significantly less: means that the amount of a product of an enzymaticreaction is reduced by more than the margin of error inherent in themeasurement technique, specifically a decrease by about 2-fold orgreater of the activity of the wild-type enzyme in the absence of theinhibitor, more specifically an decrease by about 5-fold or greater, andmost specifically an decrease by about 10-fold or greater.

Specific Binding/immunological Cross-Reactivity: An indication that twonucleic acid sequences or proteins are substantially identical is thatthe protein encoded by the first nucleic acid is immunologically crossreactive with, or specifically binds to, the protein encoded by thesecond nucleic acid. Thus, a protein is typically substantiallyidentical to a second protein, for example, where the two proteinsdiffer only by conservative substitutions. The phrase “specifically (orselectively) binds to an antibody,” or “specifically (or selectively)immunoreactive with,” when referring to a protein or peptide, refers toa binding reaction which is determinative of the presence of the proteinin the presence of a heterogeneous population of proteins and otherbiologics. Thus, under designated immunoassay conditions, the specifiedantibodies bind to a particular protein and do not bind in a significantamount to other proteins present in the sample. Specific binding to anantibody under such conditions may require an antibody that is selectedfor its specificity for a particular protein. For example, antibodiesraised to the protein with the amino acid sequence encoded by any of thenucleic acid sequences of the invention can be selected to obtainantibodies specifically immunoreactive with that protein and not withother proteins except for polymorphic variants. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassays,Western blots, or immunohistochemistry are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York “Harlow and Lane”), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity. Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays” Elsevier,New York. Generally, highly stringent hybridization and wash conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. Typically,under “stringent conditions” a probe will hybridize to its targetsubsequence, but to no other sequences.

The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe. Verystringent conditions are selected to be equal to the Tm for a particularprobe. An example of stringent hybridization conditions forhybridization of complementary nucleic acids which have more than 100complementary residues on a filter in a Southern or northern blot is 50%formamide with 1 mg of heparin at 42° C., with the hybridization beingcarried out overnight. An example of highly stringent wash conditions is0.1 5M NaCl at 72° C. for about 15 minutes. An example of stringent washconditions is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook,infra, for a description of SSC buffer). Often, a high stringency washis preceded by a low stringency wash to remove background probe signal.An example medium stringency wash for a duplex of, e.g., more than 100nucleotides, is 1×SSC at 45° C. for 15 minutes. An example lowstringency wash for a duplex of, e.g., more than 100 nucleotides, is4-6×SSC at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50nucleotides), stringent conditions typically involve salt concentrationsof less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ionconcentration (or other salts) at pH 7.0 to 8.3, and the temperature istypically at least about 30° C. Stringent conditions can also beachieved with the addition of destabilizing agents such as formamide. Ingeneral, a signal to noise ratio of 2× (or higher) than that observedfor an unrelated probe in the particular hybridization assay indicatesdetection of a specific hybridization. Nucleic acids that do nothybridize to each other under stringent conditions are stillsubstantially identical if the proteins that they encode aresubstantially identical. This occurs, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone nucleotide sequences that are homologues ofreference nucleotide sequences of the present invention: a referencenucleotide sequence specifically hybridizes to the reference nucleotidesequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at50° C. with washing in 2×SSC, 0.1% SDS at 50° C., more desirably in 7%sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. withwashing in 1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in0.5×SC, 0.1% SDS at 50° C., specifically in 7% sodium dodecyl sulfate(SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1%SDS at 50° C., more specifically in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SOS at 65° C.

A “subsequence” refers to a sequence of nucleic acids or amino acidsthat comprise a part of a longer sequence of nucleic acids or aminoacids (e.g., protein) respectively.

Substantial similarity: The term “substantial similarity” in the contextof two nucleic acid or protein sequences, refers to two or moresequences or subsequences that are substantially similar, for examplethat have 50%, specifically 60%, more specifically 70%, even morespecifically 80%, still more specifically 90%, further more specifically95%, and most specifically 99% sequence identity.

Substrate: a substrate is the molecule that an enzyme naturallyrecognizes and converts to a product in the biochemical pathway in whichthe enzyme naturally carries out its function, or is a modified versionof the molecule, which is also recognized by the enzyme and is convertedby the enzyme to a product in an enzymatic reaction similar to thenaturally-occurring reaction.

Transformation: a process for introducing heterologous DNA into a plantcell, plant tissue, or plant. Transformed plant cells, plant tissue, orplants are understood to encompass not only the end product of atransformation process, but also transgenic progeny thereof.

“Transformed,” “transgenic,” and “recombinant” refer to a host organismsuch as a bacterium or a plant into which a heterologous nucleic acidmolecule has been introduced. The nucleic acid molecule can be stablyintegrated into the genome of the host or the nucleic acid molecule canalso be present as an extrachromosomal molecule. Such anextrachromosomal molecule can be auto-replicating. Transformed cells,tissues, or plants are understood to encompass not only the end productof a transformation process, but also transgenic progeny thereof. A“non-transformed,” “non-transgenic,” or “non-recombinant” host refers toa wild-type organism, e.g., a bacterium or plant, which does not containthe heterologous nucleic acid molecule.

Viability: “viability” as used herein refers to a fitness parameter of aplant. Plants are assayed for their homozygous performance of plantdevelopment, indicating which proteins are essential for plant growth.

DETAILED DESCRIPTION OF THE INVENTION I. General Description of TraitFunctional Genomics

The goal of functional genomics is to identify genes controllingexpression of organismal phenotypes, and employs a variety ofmethodologies, including but not limited to bioinformatics, geneexpression studies, gene and gene product interactions, genetics,biochemistry and molecular genetics. For example, bioinformatics canassign function to a given gene by identifying genes in heterologousorganisms with a high degree of similarity (homology) at the amino acidor nucleotide level. Expression of a gene at the mRNA or protein levelscan assign function by linking expression of a gene to an environmentalresponse, a developmental process or a genetic (mutational) or moleculargenetic (gene overexpression or underexpression) perturbation.Expression of a gene at the mRNA level can be ascertained either alone(Northern analysis) or in concert with other genes (microarrayanalysis), whereas expression of a gene at the protein level can beascertained either alone (native or denatured protein gel or immunoblotanalysis) or in concert with other genes (proteomic analysis). Knowledgeof protein/protein and protein/DNA interactions can assign function byidentifying proteins and nucleic acid sequences acting together in thesame biological process. Genetics can assign function to a gene bydemonstrating that DNA lesions (mutations) in the gene have aquantifiable effect on the organism, including but not limited to: itsdevelopment; hormone biosynthesis and response; growth and growth habit(plant architecture); mRNA expression profiles; protein expressionprofiles; ability to resist diseases; tolerance of abiotic stresses;ability to acquire nutrients; photosynthetic efficiency; altered primaryand secondary metabolism; and the composition of various plant organs.Biochemistry can assign function by demonstrating that the proteinencoded by the gene, typically when expressed in a heterologousorganism, possesses a certain enzymatic activity, alone or incombination with other proteins. Molecular genetics can assign functionby overexpressing or underexpressing the gene in the native plant or inheterologous organisms, and observing quantifiable effects as describedin functional assignment by genetics above. In functional genomics, anyor all of these approaches are utilized, often in concert, to assigngenes to functions across any of a number of organismal phenotypes.

It is recognized by those skilled in the art that these differentmethodologies can each provide data as evidence for the function of aparticular gene, and that such evidence is stronger with increasingamounts of data used for functional assignment: specifically from asingle methodology, more specifically from two methodologies, and evenmore specifically from more than two methodologies. In addition, thoseskilled in the art are aware that different methodologies can differ inthe strength of the evidence for the assignment of gene function.Typically, but not always, a datum of biochemical, genetic and moleculargenetic evidence is considered stronger than a datum of bioinformatic orgene expression evidence. Finally, those skilled in the art recognizethat, for different genes, a single datum from a single methodology candiffer in terms of the strength of the evidence provided by eachdistinct datum for the assignment of the function of these differentgenes.

The objective of crop trait functional genomics is to identify croptrait genes, i.e. genes capable of conferring useful agronomic traits incrop plants. Such agronomic traits include, but are not limited to:enhanced yield, whether in quantity or quality; enhanced nutrientacquisition and enhanced metabolic efficiency; enhanced or alterednutrient composition of plant tissues used for food, feed, fiber orprocessing; enhanced utility for agricultural or industrial processing;enhanced resistance to plant diseases; enhanced tolerance of adverseenvironmental conditions (abiotic stresses) including but not limited todrought, excessive cold, excessive heat, or excessive soil salinity orextreme acidity or alkalinity; and alterations in plant architecture ordevelopment, including changes in developmental timing. The deploymentof such identified trait genes by either transgenic or non-transgenicmeans could materially improve crop plants for the benefit ofagriculture.

Cereals are the most important crop plants on the planet, in terms ofboth human and animal consumption. Genomic synteny (conservation of geneorder within large chromosomal segments) is observed in rice, maize,wheat, barley, rye, oats and other agriculturally important monocots,which facilitates the mapping and isolation of orthologous genes fromdiverse cereal species based on the sequence of a single cereal gene.Rice has the smallest (˜420 Mb) genome among the cereal grains, and hasrecently been a major focus of public and private genomic and ESTsequencing efforts.

To identify crop trait genes in the rice [wheat] genome controlling[trait], genes from the rice draft genome sequence [wheat EST databases]were prioritized based on one or more functional genomic methodologies.For example, genome-wide expression studies of rice plants infected withrice blast fungus (Magnaporthe grisea) were used to prioritize candidategenes controlling disease resistance. Full-length and partial cDNAs ofrice trait gene candidates could then be predicted based on analysis ofthe rice whole-genome sequence, and isolated by designing and usingprimers for PCR amplification using a commercially available PCRprimer-picking program. Primers were used for PCR amplification offull-length or partial cDNAs from rice cDNA libraries or first-strandcDNA. cDNA clones resulting from either approach were used for theconstruction of vectors designed for altering expression of these genesin transgenic plants using plant molecular genetic methodologies, whichare described in detail below. Alteration of plant phenotype throughoverexpression or underexpression of key trait genes in transgenicplants is a robust and established method for assigning functions toplant genes. Assays to identify transgenic plants with alterations intraits of interest are to be used to unambiguously assign the utility ofthese genes for the improvement of rice, and by extension, othercereals, either by transgenic or classical breeding methods.

II. Identifying, Cloning and Sequencing cDNAs

The cloning and sequencing of the cDNAs of the present invention aredescribed in Example 1.

The isolated nucleic acids and proteins of the present invention areusable over a range of plants, monocots and dicots, in particularmonocots such as rice, wheat, barley and maize. In a more specificembodiment, the monocot is a cereal. In a more specific embodiment, thecereal may be, for example, maize, wheat, barley, oats, rye, millet,sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax,gramma grass, Tripsacum sp., or teosinte. In a most specific embodiment,the cereal is rice. Other plants genera include, but are not limited to,Cucurbita, Rosa, Vitis, Juglans, Gragaria, Lotus, Medicago, Onobrychis,Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus,Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum.

The present invention also provides a method of genotyping a plant orplant part comprising a nucleic acid molecule of the present invention.Optionally, the plant is a monocot such as, but not limited rice orwheat. Genotyping provides a means of distinguishing homologs of achromosome pari and can be used to differentiate segregants in a plantpopulation. Molecular marker methods can be used in phylogeneticstudies, characterizing genetic relationships among crop varieties,identifying crosses or somatic hybrids, localizing chromosomeal segmentsaffecting mongenic traits, map based cloning, and the study ofquantitative inheritance (see Plant Molecular Biology: A LaboratoryManual, Chapter 7, Clark ed., Springer-Verlag, Berlin 1997; Paterson, A.H., “The DNA Revolution”, chapter 2 in Genome Mapping in Plants,Paterson, A. H. ed., Academic Press/R.G. Lands Co., Austin, Tex. 1996).

The method of genotyping may employ any number of molecular markeranalytical techniques such as, but not limited to, restriction lengthpolymorphisms (RFLPs). As is well known in the art, RFLPs are producedby differences in the DNA restriction fragment lengths resulting fromnucleotide differences between alleles of the same gene. Thus, thepresent invention provides a method of following segregation of a geneor nucleic acid of the present invention or chromosomal sequencesgenetically linked by using RFLP analysis. Linked chromosomal sequencesare within 50 centiMorgans (50 cM), within 40 or 30 cM, specificallywithin 20 or 10 cM, more specifically within 5, 3, 2, or 1 cM of thenucleic acid of the invention.

III. Traits of Interest

The present invention encompasses the identification and isolation ofpolynucleotides encoding proteins involved in sugar sensing and,ultimately, in nitrogen uptake and carbon metabolism. Altering theexpression of genes related to these traits can be used to improve ormodify plants and/or grain, as desired. Examples describe the isolatedgenes of interest and methods of analyzing the alteration of expressionand their effects on the plant characteristics.

One aspect of the present invention provides compositions and methodsfor altering (i.e. increasing or decreasing) the level of nucleic acidmolecules and polypeptides of the present invention in plants. Inparticular, the nucleic acid molecules and polypeptides of the inventionare expressed constitutively, temporally or spatially, e.g. atdevelopmental stages, in certain tissues, and/or quantities, which areuncharacteristic of non-recombinantly engineered plants. Therefore, thepresent invention provides utility in such exemplary applications asaltering the specified characteristics identified above.

VI. Controlling Gene Expression in Transgenic Plants

The invention further relates to transformed cells comprising thenucleic acid molecules, transformed plants, seeds, and plant parts, andmethods of modifying phenotypic traits of interest by altering theexpression of the genes of the invention.

A. Modification of Coding Sequences and Adjacent Sequences

The transgenic expression in plants of genes derived from heterologoussources may involve the modification of those genes to achieve andoptimize their expression in plants. In particular, bacterial ORFs whichencode separate enzymes but which are encoded by the same transcript inthe native microbe are best expressed in plants on separate transcripts.To achieve this, each microbial ORF is isolated individually and clonedwithin a cassette which provides a plant promoter sequence at the 5′ endof the ORF and a plant transcriptional terminator at the 3′ end of theORF. The isolated ORF sequence specifically includes the initiating ATGcodon and the terminating STOP codon but may include additional sequencebeyond the initiating ATG and the STOP codon. In addition, the ORF maybe truncated, but still retain the required activity; for particularlylong ORFs, truncated versions which retain activity may be preferablefor expression in transgenic organisms. By “plant promoter” and “planttranscriptional terminator” it is intended to mean promoters andtranscriptional terminators that operate within plant cells. Thisincludes promoters and transcription terminators that may be derivedfrom non-plant sources such as viruses (an example is the CauliflowerMosaic Virus).

In some cases, modification to the ORF coding sequences and adjacentsequence is not required. It is sufficient to isolate a fragmentcontaining the ORF of interest and to insert it downstream of a plantpromoter. For example, Gaffney et al. (Science 261: 754-756 (1993)) haveexpressed the Pseudomonas nahG gene in transgenic plants under thecontrol of the CaMV 35S promoter and the CaMV tml terminatorsuccessfully without modification of the coding sequence and withnucleotides of the Pseudomonas gene upstream of the ATG still attached,and nucleotides downstream of the STOP codon still attached to the nahGORF. Specifically, as little adjacent microbial sequence as possibleshould be left attached upstream of the ATG and downstream of the STOPcodon. In practice, such construction may depend on the availability ofrestriction sites.

In other cases, the expression of genes derived from microbial sourcesmay provide problems in expression. These problems have been wellcharacterized in the art and are particularly common with genes derivedfrom certain sources such as Bacillus. These problems may apply to thenucleotide sequence of this invention and the modification of thesegenes can be undertaken using techniques now well known in the art. Thefollowing problems may be encountered:

1. Codon Usage.

The specific codon usage in plants differs from the specific codon usagein certain microorganisms. Comparison of the usage of codons within acloned microbial ORF to usage in plant genes (and in particular genesfrom the target plant) will enable an identification of the codonswithin the ORF that should specifically be changed. Typically plantevolution has tended towards a strong preference of the nucleotides Cand G in the third base position of monocotyledons, whereas dicotyledonsoften use the nucleotides A or T at this position. By modifying a geneto incorporate specific codon usage for a particular target transgenicspecies, many of the problems described below for GC/AT content andillegitimate splicing will be overcome.

2. GC/AT Content.

Plant genes typically have a GC content of more than 35%. ORF sequenceswhich are rich in A and T nucleotides can cause several problems inplants. Firstly, motifs of ATTTA are believed to cause destabilizationof messages and are found at the 3′ end of many short-lived mRNAs.Secondly, the occurrence of polyadenylation signals such as AATAAA atinappropriate positions within the message is believed to causepremature truncation of transcription. In addition, monocotyledons mayrecognize AT-rich sequences as splice sites (see below).

3. Sequences Adjacent to the Initiating Methionine.

Plants differ from microorganisms in that their messages do not possessa defined ribosome-binding site. Rather, it is believed that ribosomesattach to the 5′ end of the message and scan for the first available ATGat which to start translation. Nevertheless, it is believed that thereis a preference for certain nucleotides adjacent to the ATG and thatexpression of microbial genes can be enhanced by the inclusion of aeukaryotic consensus translation initiator at the ATG. Clontech(1993/1994 catalog, page 210, incorporated herein by reference) havesuggested one sequence as a consensus translation initiator for theexpression of the E. coli uidA gene in plants. Further, Joshi (N.A.R.15: 6643-6653 (1987), incorporated herein by reference) has comparedmany plant sequences adjacent to the ATG and suggests another consensussequence. In situations where difficulties are encountered in theexpression of microbial ORFs in plants, inclusion of one of thesesequences at the initiating ATG may improve translation. In such casesthe last three nucleotides of the consensus may not be appropriate forinclusion in the modified sequence due to their modification of thesecond AA residue. Specific sequences adjacent to the initiatingmethionine may differ between different plant species. A survey of 14maize genes located in the GenBank database provided the followingresults:

Position Before the Initiating ATG in 14 Maize Genes:

−10 −9 −8 −7 −6 −5 −4 −3 −2 −1 C 3 8 4 6 2 5 6 0 10 7 T 3 0 3 4 3 2 1 11 0 A 2 3 1 4 3 2 3 7 2 3 G 6 3 6 0 6 5 4 6 1 5This analysis can be done for the desired plant species into which thenucleotide sequence is being incorporated, and the sequence adjacent tothe ATG modified to incorporate the specific nucleotides.

4. Removal of Illegitimate Splice Sites.

Genes cloned from non-plant sources and not optimized for expression inplants may also contain motifs which may be recognized in plants as 5′or 3′ splice sites, and be cleaved, thus generating truncated or deletedmessages. These sites can be removed using the techniques well known inthe art.

Techniques for the modification of coding sequences and adjacentsequences are well known in the art. In cases where the initialexpression of a microbial ORF is low and it is deemed appropriate tomake alterations to the sequence as described above, then theconstruction of synthetic genes can be accomplished according to methodswell known in the art. These are, for example, described in thepublished patent disclosures EP 0 385 962 (to Monsanto), EP 0 359 472(to Lubrizol) and WO 93/07278 (to Ciba-Geigy), all of which areincorporated herein by reference. In most cases it is preferable toassay the expression of gene constructions using transient assayprotocols (which are well known in the art) prior to their transfer totransgenic plants.

B. Construction of Plant Expression Cassettes

Coding sequences intended for expression in transgenic plants are firstassembled in expression cassettes behind a suitable promoter expressiblein plants. The expression cassettes may also comprise any furthersequences required or selected for the expression of the transgene. Suchsequences include, but are not restricted to, transcription terminators,extraneous sequences to enhance expression such as introns, vitalsequences, and sequences intended for the targeting of the gene productto specific organelles and cell compartments. These expression cassettescan then be easily transferred to the plant transformation vectorsdescribed below. The following is a description of various components oftypical expression cassettes.

1. Promoters

The selection of the promoter used in expression cassettes willdetermine the spatial and temporal expression pattern of the transgenein the transgenic plant. Selected promoters will express transgenes inspecific cell types (such as leaf epidermal cells, mesophyll cells, rootcortex cells) or in specific tissues or organs (roots, leaves orflowers, for example) and the selection will reflect the desiredlocation of accumulation of the gene product. Alternatively, theselected promoter may drive expression of the gene under variousinducing conditions. Promoters vary in their strength, i.e., ability topromote transcription. Depending upon the host cell system utilized, anyone of a number of suitable promoters can be used, including the gene'snative promoter. The following are non-limiting examples of promotersthat may be used in expression cassettes.

a. Constitutive Expression, the Ubiquitin Promoter:

Ubiquitin is a gene product known to accumulate in many cell types andits promoter has been cloned from several species for use in transgenicplants (e.g. sunflower—Binet et al. Plant Science 79: 87-94 (1991);maize—Christensen et al. Plant Molec. Biol. 12: 619-632 (1989); andArabidopsis—Callis et al., J. Biol. Chem. 265:12486-12493 (1990) andNorris et al., Plant Mol. Biol. 21:895-906 (1993)). The maize ubiquitinpromoter has been developed in transgenic monocot systems and itssequence and vectors constructed for monocot transformation aredisclosed in the patent publication EP 0 342 926 (to Lubrizol) which isherein incorporated by reference. Taylor et al. (Plant Cell Rep. 12:491-495 (1993)) describe a vector (pAHC25) that comprises the maizeubiquitin promoter and first intron and its high activity in cellsuspensions of numerous monocotyledons when introduced viamicroprojectile bombardment. The Arabidopsis ubiquitin promoter is idealfor use with the nucleotide sequences of the present invention. Theubiquitin promoter is suitable for gene expression in transgenic plants,both monocotyledons and dicotyledons. Suitable vectors are derivativesof pAHC25 or any of the transformation vectors described in thisapplication, modified by the introduction of the appropriate ubiquitinpromoter and/or intron sequences.

b. Constitutive Expression, the CaMV 35S Promoter:

Construction of the plasmid pCGN1761 is described in the publishedpatent application EP 0 392 225 (Example 23), which is herebyincorporated by reference. pCGN1761 contains the “double” CaMV 35Spromoter and the tml transcriptional terminator with a unique EcoRI sitebetween the promoter and the terminator and has a pUC-type backbone. Aderivative of pCGN1761 is constructed which has a modified polylinkerwhich includes NotI and XhoI sites in addition to the existing EcoRIsite. This derivative is designated pCGN1761ENX. pCGN1761ENX is usefulfor the cloning of cDNA sequences or coding sequences (includingmicrobial ORF sequences) within its polylinker for the purpose of theirexpression under the control of the 35S promoter in transgenic plants.The entire 35S promoter-coding sequence-tml terminator cassette of sucha construction can be excised by HindIII, SphI, SalI, and XbaI sites 5′to the promoter and XbaI, BamHI and BglI sites 3′ to the terminator fortransfer to transformation vectors such as those described below.Furthermore, the double 35S promoter fragment can be removed by 5′excision with HindIII, SphI, SalI, XbaI, or PstI, and 3′ excision withany of the polylinker restriction sites (EcoRI, NotI or XhoI) forreplacement with another promoter. If desired, modifications around thecloning sites can be made by the introduction of sequences that mayenhance translation. This is particularly useful when overexpression isdesired. For example, pCGN1761ENX may be modified by optimization of thetranslational initiation site as described in Example 37 of U.S. Pat.No. 5,639,949, incorporated herein by reference.

c. Constitutive Expression, the Actin Promoter:

Several isoforms of actin are known to be expressed in most cell typesand consequently the actin promoter is a good choice for a constitutivepromoter. In particular, the promoter from the rice Actl gene has beencloned and characterized (McElroy et al., Plant Cell 2: 163-171 (1990)).A 1.3 kb fragment of the promoter was found to contain all theregulatory elements required for expression in rice protoplasts.Furthermore, numerous expression vectors based on the Actl promoter havebeen constructed specifically for use in monocotyledons (McElroy et al.Mol. Gen. Genet. 231: 150-160 (1991)). These incorporate the Actl-intron1, Adhl 5′ flanking sequence and Adhl-intron 1 (from the maize alcoholdehydrogenase gene) and sequence from the CaMV 35S promoter. Vectorsshowing highest expression were fusions of 35S and Actl intron or theActl 5′ flanking sequence and the Actl intron. Optimization of sequencesaround the initiating ATG (of the GUS reporter gene) also enhancedexpression. The promoter expression cassettes described by McElroy etal. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified forgene expression and are particularly suitable for use inmonocotyledonous hosts. For example, promoter-containing fragments isremoved from the McElroy constructions and used to replace the double35S promoter in pCGN1761ENX, which is then available for the insertionof specific gene sequences. The fusion genes thus constructed can thenbe transferred to appropriate transformation vectors. In a separatereport, the rice Actl promoter with its first intron has also been foundto direct high expression in cultured barley cells (Chibbar et al. PlantCell Rep. 12: 506-509 (1993)).

d. Inducible Expression, PR-1 Promoters:

The double 35S promoter in pCGN1761ENX may be replaced with any otherpromoter of choice that will result in suitably high expression levels.By way of example, one of the chemically regulatable promoters describedin U.S. Pat. No. 5,614,395, such as the tobacco PR-1a promoter, mayreplace the double 35S promoter. Alternately, the Arabidopsis PR-1promoter described in Lebel et al., Plant J. 16:223-233 (1998) may beused. The promoter of choice is specifically excised from its source byrestriction enzymes, but can alternatively be PCR-amplified usingprimers that carry appropriate terminal restriction sites. ShouldPCR-amplification be undertaken, the promoter should be re-sequenced tocheck for amplification errors after the cloning of the amplifiedpromoter in the target vector. The chemically/pathogen regulatabletobacco PR-1a promoter is cleaved from plasmid pCIB1004 (forconstruction, see example 21 of EP 0 332 104, which is herebyincorporated by reference) and transferred to plasmid pCGN1761ENX (Ukneset al., Plant Cell 4: 645-656 (1992)). pCIB1004 is cleaved with NcoI andthe resultant 3′ overhang of the linearized fragment is rendered bluntby treatment with T4 DNA polymerase. The fragment is then cleaved withHindIII and the resultant PR-1a promoter-containing fragment is gelpurified and cloned into pCGN1761ENX from which the double 35S promoterhas been removed. This is accomplished by cleavage with XhoI andblunting with T4 polymerase, followed by cleavage with HindIII, andisolation of the larger vector-terminator containing fragment into whichthe pCIB1004 promoter fragment is cloned. This generates a pCGN1761ENXderivative with the PR-1a promoter and the tml terminator and anintervening polylinker with unique EcoRI and NotI sites. The selectedcoding sequence can be inserted into this vector, and the fusionproducts (i.e. promoter-gene-terminator) can subsequently be transferredto any selected transformation vector, including those described infra.Various chemical regulators may be employed to induce expression of theselected coding sequence in the plants transformed according to thepresent invention, including the benzothiadiazole, isonicotinic acid,and salicylic acid compounds disclosed in U.S. Pat. Nos. 5,523,311 and5,614,395.

e. Inducible Expression, an Ethanol-Inducible Promoter:

A promoter inducible by certain alcohols or ketones, such as ethanol,may also be used to confer inducible expression of a coding sequence ofthe present invention. Such a promoter is for example the alcA genepromoter from Aspergillus nidulans (Caddick et al. (1998) Nat.Biotechnol 16:177-180). In A. nidulans, the alcA gene encodes alcoholdehydrogenase 1, the expression of which is regulated by the AlcRtranscription factors in presence of the chemical inducer. For thepurposes of the present invention, the CAT coding sequences in plasmidpalcA:CAT comprising a alcA gene promoter sequence fused to a minimal35S promoter (Caddick et al. (1998) Nat. Biotechnol 16:177-180) arereplaced by a coding sequence of the present invention to form anexpression cassette having the coding sequence under the control of thealcA gene promoter. This is carried out using methods well known in theart.

f. Inducible Expression, a Glucocorticoid-Inducible Promoter:

Induction of expression of a nucleic acid sequence of the presentinvention using systems based on steroid hormones is also contemplated.For example, a glucocorticoid-mediated induction system is used (Aoyamaand Chua (1997) The Plant Journal 11: 605-612) and gene expression isinduced by application of a glucocorticoid, for example a syntheticglucocorticoid, specifically dexamethasone, specifically at aconcentration ranging from 0.1 mM to 1 mM, more specifically from 10 mMto 100 mM. For the purposes of the present invention, the luciferasegene sequences are replaced by a nucleic acid sequence of the inventionto form an expression cassette having a nucleic acid sequence of theinvention under the control of six copies of the GAL4 upstreamactivating sequences fused to the 35S minimal promoter. This is carriedout using methods well known in the art. The trans-acting factorcomprises the GAL4 DNA-binding domain (Keegan et al. (1986) Science 231:699-704) fused to the transactivating domain of the herpes viral proteinVP16 (Triezenberg et al. (1988) Genes Devel. 2: 718-729) fused to thehormone-binding domain of the rat glucocorticoid receptor (Picard et al.(1988) Cell 54: 1073-1080). The expression of the fusion protein iscontrolled either by a promoter known in the art or described here. Thisexpression cassette is also comprised in the plant comprising a nucleicacid sequence of the invention fused to the 6×GAL4/minimal promoter.Thus, tissue- or organ-specificity of the fusion protein is achievedleading to inducible tissue- or organ-specificity of the insecticidaltoxin.

g. Root Specific Expression:

Another pattern of gene expression is root expression. A suitable rootpromoter is the promoter of the maize metallothionein-like (MTL) genedescribed by de Framond (FEBS 290: 103-106 (1991)) and also in U.S. Pat.No. 5,466,785, incorporated herein by reference. This “MTL” promoter istransferred to a suitable vector such as pCGN1761ENX for the insertionof a selected gene and subsequent transfer of the entirepromoter-gene-terminator cassette to a transformation vector ofinterest.

h. Wound-Inducible Promoters:

Wound-inducible promoters may also be suitable for gene expression.Numerous such promoters have been described (e.g. Xu et al. Plant Molec.Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989),Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al.Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191-201(1993)) and all are suitable for use with the instant invention.Logemann et al. describe the 5′ upstream sequences of the dicotyledonouspotato wunl gene. Xu et al. show that a wound-inducible promoter fromthe dicotyledon potato (pin2) is active in the monocotyledon rice.Further, Rohrmeier & Lehle describe the cloning of the maize Wipl cDNAwhich is wound induced and which can be used to isolate the cognatepromoter using standard techniques. Similar, Firek et al. and Warner etal. have described a wound-induced gene from the monocotyledon Asparagusofficinalis, which is expressed at local wound and pathogen invasionsites. Using cloning techniques well known in the art, these promoterscan be transferred to suitable vectors, fused to the genes pertaining tothis invention, and used to express these genes at the sites of plantwounding.

i. Pith-Specific Expression:

Patent Application WO 93/07278, which is herein incorporated byreference, describes the isolation of the maize trpA gene, which ispreferentially expressed in pith cells. The gene sequence and promoterextending up to −1726 bp from the start of transcription are presented.Using standard molecular biological techniques, this promoter, or partsthereof can be transferred to a vector such as pCGN1761 where it canreplace the 35S promoter and be used to drive the expression of aforeign gene in a pith-specific manner. In fact, fragments containingthe pith-specific promoter or parts thereof can be transferred to anyvector and modified for utility in transgenic plants.

j. Leaf-Specific Expression:

A maize gene encoding phosphoenol carboxylase (PEPC) has been describedby Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Usingstandard molecular biological techniques the promoter for this gene canbe used to drive the expression of any gene in a leaf-specific manner intransgenic plants.

k. Pollen-Specific Expression:

WO 93/07278 describes the isolation of the maize calcium-dependentprotein kinase (CDPK) gene which is expressed in pollen cells. The genesequence and promoter extend up to 1400 bp from the start oftranscription. Using standard molecular biological techniques, thispromoter or parts thereof, can be transferred to a vector such aspCGN1761 where it can replace the 35S promoter and be used to drive theexpression of a nucleic acid sequence of the invention in apollen-specific manner.

2. Transcriptional Terminators

A variety of transcriptional terminators are available for use inexpression cassettes. These are responsible for the termination oftranscription beyond the transgene and correct mRNA polyadenylation.Appropriate transcriptional terminators are those that are known tofunction in plants and include the CaMV 35S terminator, the tmlterminator, the nopaline synthase terminator and the pea rbcS E9terminator. These can be used in both monocotyledons and dicotyledons.In addition, a gene's native transcription terminator may be used.

3. Sequences for the Enhancement or Regulation of Expression

Numerous sequences have been found to enhance gene expression fromwithin the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize Adhl gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200(1987)). In the same experimental system, the intron from the maizebronze1 gene had a similar effect in enhancing expression. Intronsequences have been routinely incorporated into plant transformationvectors, typically within the non-translated leader.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are particularly effectivein dicotyledonous cells. Specifically, leader sequences from TobaccoMosaic Virus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus(MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effectivein enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15;8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).Other leader sequences known in the art include but are not limited to:picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. PNAS USA86:6126-6130 (1989)); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20); human immunoglobulin heavy-chainbinding protein (BiP) leader, (Macejak, D. G., and Sarnow, P., Nature353: 90-94 (1991); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L.,Nature 325:622-625 (1987); tobacco mosaic virus leader (TMV), (Gallie,D. R. et al., Molecular Biology of RNA, pages 237-256 (1989); and MaizeChlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al., Virology81:382-385 (1991). See also, Della-Cioppa et al., Plant Physiology.84:965-968 (1987).

In addition to incorporating one or more of the aforementioned elementsinto the 5′ regulatory region of a target expression cassette of theinvention, other elements peculiar to the target expression cassette mayalso be incorporated. Such elements include but are not limited to aminimal promoter. By minimal promoter it is intended that the basalpromoter elements are inactive or nearly so without upstream activation.Such a promoter has low background activity in plants when there is notransactivator present or when enhancer or response element bindingsites are absent. One minimal promoter that is particularly useful fortarget genes in plants is the Bz1 minimal promoter, which is obtainedfrom the bronze1 gene of maize. The Bz1 core promoter is obtained fromthe “myc” mutant Bz1-luciferase construct pBz1LucR98 via cleavage at theNheI site located at −53 to −58. Roth et al., Plant Cell 3: 317 (1991).The derived Bz1 core promoter fragment thus extends from −53 to +227 andincludes the Bz1 intron-1 in the 5′ untranslated region. Also useful forthe invention is a minimal promoter created by use of a synthetic TATAelement, The TATA element allows recognition of the promoter by RNApolymerase factors and confers a basal level of gene expression in theabsence of activation (see generally, Mukumoto (1993) Plant Mol Biol 23;995-1003; Green (2000) Trends Biochem Sci 25: 59-63)

4. Targeting of the Gene Product within the Cell

Various mechanisms for targeting gene products are known to exist inplants and the sequences controlling the functioning of these mechanismshave been characterized in some detail. For example, the targeting ofgene products to the chloroplast is controlled by a signal sequencefound at the amino terminal end of various proteins which is cleavedduring chloroplast import to yield the mature protein (e.g. Comai et al.J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can befused to heterologous gene products to effect the import of heterologousproducts into the chloroplast (van den Broeck, et al. Nature 313:358-363 (1985)). DNA encoding for appropriate signal sequences can beisolated from the 5′ end of the cDNAs encoding the RUBISCO protein, theCAB protein, the EPSP synthase enzyme, the GS2 protein and many otherproteins which are known to be chloroplast localized. See also, thesection entitled “Expression With Chloroplast Targeting” in Example 37of U.S. Pat. No. 5,639,949.

Other gene products are localized to other organelles such as themitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol.13: 411-418 (1989)). The cDNAs encoding these products can also bemanipulated to effect the targeting of heterologous gene products tothese organelles. Examples of such sequences are the nuclear-encodedATPases and specific aspartate amino transferase isoforms formitochondria. Targeting cellular protein bodies has been described byRogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

In addition, sequences have been characterized which cause the targetingof gene products to other cell compartments. Amino terminal sequencesare responsible for targeting to the ER, the apoplast, and extracellularsecretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783(1990)). Additionally, amino terminal sequences in conjunction withcarboxy terminal sequences are responsible for vacuolar targeting ofgene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).

By the fusion of the appropriate targeting sequences described above totransgene sequences of interest it is possible to direct the transgeneproduct to any organelle or cell compartment. For chloroplast targeting,for example, the chloroplast signal sequence from the RUBISCO gene, theCAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame tothe amino terminal ATG of the transgene. The signal sequence selectedshould include the known cleavage site, and the fusion constructedshould take into account any amino acids after the cleavage site whichare required for cleavage. In some cases this requirement may befulfilled by the addition of a small number of amino acids between thecleavage site and the transgene ATG or, alternatively, replacement ofsome amino acids within the transgene sequence. Fusions constructed forchloroplast import can be tested for efficacy of chloroplast uptake byin vitro translation of in vitro transcribed constructions followed byin vitro chloroplast uptake using techniques described by Bartlett etal. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology,Elsevier pp 1081-1091 (1982) and Wasmann et al. Mol. Gen. Genet. 205:446-453 (1986). These construction techniques are well known in the artand are equally applicable to mitochondria and peroxisomes.

The above-described mechanisms for cellular targeting can be utilizednot only in conjunction with their cognate promoters, but also inconjunction with heterologous promoters so as to effect a specificcell-targeting goal under the transcriptional regulation of a promoterthat has an expression pattern different to that of the promoter fromwhich the targeting signal derives.

C. Construction of Plant Transformation Vectors

Numerous transformation vectors available for plant transformation areknown to those of ordinary skill in the plant transformation arts, andthe genes pertinent to this invention can be used in conjunction withany such vectors. The selection of vector will depend upon the specifictransformation technique and the target species for transformation. Forcertain target species, different antibiotic or herbicide selectionmarkers may be specific. Selection markers used routinely intransformation include the nptII gene, which confers resistance tokanamycin and related antibiotics (Messing & Vierra. Gene 19; 259-268(1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, whichconfers resistance to the herbicide phosphinothricin (White et al.,Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet. 79:625-631 (1990)), the hph gene, which confers resistance to theantibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4:2929-2931), and the dhfr gene, which confers resistance to methatrexate(Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), the EPSPS gene, whichconfers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and5,188,642), and the mannose-6-phosphate isomerase gene, which providesthe ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and5,994,629).

1. Vectors Suitable for Agrobacterium Transformation

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)).Below, the construction of two typical vectors suitable forAgrobacterium transformation is described.

a. pCIB200 and pCIB2001:

The binary vectors pCIB200 and pCIB2001 are used for the construction ofrecombinant vectors for use with Agrobacterium and are constructed inthe following manner. pTJS75kan is created by NarI digestion of pTJS75(Schmidhauser & Helinski, J. Bacteriol. 164: 446-455 (1985)) allowingexcision of the tetracycline-resistance gene, followed by insertion ofan AccI fragment from pUC4K carrying an NPTII (Messing & Vierra, Gene19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride etal., Plant Molecular Biology 14: 266-276 (1990)). XhoI linkers areligated to the EcoRV fragment of PCIB7 which contains the left and rightT-DNA borders, a plant selectable nos/nptII chimeric gene and the pUCpolylinker (Rothstein et al., Gene 53: 153-161 (1987)), and theXhoI-digested fragment are cloned into SalI-digested pTJS75kan to createpCIB200 (see also EP 0 332 104, example 19). pCIB200 contains thefollowing unique polylinker restriction sites: EcoRI, SstI, KpnI, BglII,XbaI, and SalI. pCIB2001 is a derivative of pCIB200 created by theinsertion into the polylinker of additional restriction sites. Uniquerestriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI,BglII, XbaI, SalI, MluI, BclI, AvrlI ApaI, HpaI, and StuI. pCIB2001, inaddition to containing these unique restriction sites also has plant andbacterial kanamycin selection, left and right T-DNA borders forAgrobacterium-mediated transformation, the RK2-derived trfA function formobilization between E. coli and other hosts, and the OriT and OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

b. pCIB10 and Hygromycin Selection Derivatives Thereof:

The binary vector pCIB10 contains a gene encoding kanamycin resistancefor selection in plants and T-DNA right and left border sequences andincorporates sequences from the wide host-range plasmid pRK252 allowingit to replicate in both E. coli and Agrobacterium. Its construction isdescribed by Rothstein et al. (Gene 53: 153-161 (1987)). Variousderivatives of pCIB10 are constructed which incorporate the gene forhygromycin B phosphotransferase described by Gritz et al. (Gene 25:179-188 (1983)). These derivatives enable selection of transgenic plantcells on hygromycin only (pCIB743), or hygromycin and kanamycin(pCIB715, pCIB717).

2. Vectors Suitable for Non-Agrobacterium Transformation

Transformation without the use of Agrobacterium tumefaciens circumventsthe requirement for T-DNA sequences in the chosen transformation vectorand consequently vectors lacking these sequences can be utilized inaddition to vectors such as the ones described above which contain T-DNAsequences. Transformation techniques that do not rely on Agrobacteriuminclude transformation via particle bombardment, protoplast uptake (e.g.PEG and electroporation) and microinjection. The choice of vectordepends largely on the specific selection for the species beingtransformed. Below, the construction of typical vectors suitable fornon-Agrobacterium transformation is described.

a. pCIB3064:

pCIB3064 is a pUC-derived vector suitable for direct gene transfertechniques in combination with selection by the herbicide basta (orphosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoterin operational fusion to the E. coli GUS gene and the CaMV 35Stranscriptional terminator and is described in the PCT publishedapplication WO 93/07278. The 35S promoter of this vector contains twoATG sequences 5′ of the start site. These sites are mutated usingstandard PCR techniques in such a way as to remove the ATGs and generatethe restriction sites SspI and PvuII. The new restriction sites are 96and 37 bp away from the unique SalI site and 101 and 42 bp away from theactual start site. The resultant derivative of pCIB246 is designatedpCIB3025. The GUS gene is then excised from pCIB3025 by digestion withSalI and SacI, the termini rendered blunt and religated to generateplasmid pCIB3060. The plasmid pJIT82 is obtained from the John InnesCentre, Norwich and the a 400 bp SmaI fragment containing the bar genefrom Streptomyces viridochromogenes is excised and inserted into theHpaI site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). Thisgenerated pCIB3064, which comprises the bar gene under the control ofthe CaMV 35S promoter and terminator for herbicide selection, a gene forampicillin resistance (for selection in E. coli) and a polylinker withthe unique sites SphI, PstI, HindIII, and BamHI. This vector is suitablefor the cloning of plant expression cassettes containing their ownregulatory signals.

b. pSOG19 and pSOG35:

pSOG35 is a transformation vector that utilizes the E. coli genedihydrofolate reductase (DFR) as a selectable marker conferringresistance to methotrexate. PCR is used to amplify the 35S promoter(−800 bp), intron 6 from the maize Adh1 gene (−550 bp) and 18 bp of theGUS untranslated leader sequence from pSOG10. A 250-bp fragment encodingthe E. coli dihydrofolate reductase type II gene is also amplified byPCR and these two PCR fragments are assembled with a SacI-PstI fragmentfrom pB1221 (Clontech) which comprises the pUC19 vector backbone and thenopaline synthase terminator Assembly of these fragments generatespSOG19 which contains the 35S promoter in fusion with the intron 6sequence, the GUS leader, the DHFR gene and the nopaline synthaseterminator. Replacement of the GUS leader in pSOG19 with the leadersequence from Maize Chlorotic Mottle Virus (MCMV) generates the vectorpSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistanceand have HindIII, SphI, PstI and EcoRI sites available for the cloningof foreign substances.

3. Vector Suitable for Chloroplast Transformation

For expression of a nucleotide sequence of the present invention inplant plastids, plastid transformation vector pPH143 (WO 97/32011,example 36) is used. The nucleotide sequence is inserted into pPH143thereby replacing the PROTOX coding sequence. This vector is then usedfor plastid transformation and selection of transformants forspectinomycin resistance. Alternatively, the nucleotide sequence isinserted in pPH143 so that it replaces the aadH gene. In this case,transformants are selected for resistance to PROTOX inhibitors.

D Transformation

Once a nucleic acid sequence of the invention has been cloned into anexpression system, it is transformed into a plant cell. The receptor andtarget expression cassettes of the present invention can be introducedinto the plant cell in a number of art-recognized ways. Methods forregeneration of plants are also well known in the art. For example, Tiplasmid vectors have been utilized for the delivery of foreign DNA, aswell as direct DNA uptake, liposomes, electroporation, microinjection,and microprojectiles. In addition, bacteria from the genus Agrobacteriumcan be utilized to transform plant cells. Below are descriptions ofrepresentative techniques for transforming both dicotyledonous andmonocotyledonous plants, as well as a representative plastidtransformation technique.

1. Transformation of Dicotyledons

Transformation techniques for dicotyledons are well known in the art andinclude Agrobacterium-based techniques and techniques that do notrequire Agrobacterium. Non-Agrobacterium techniques involve the uptakeof exogenous genetic material directly by protoplasts or cells. This canbe accomplished by PEG or electroporation mediated uptake, particlebombardment-mediated delivery, or microinjection. Examples of thesetechniques are described by Paszkowski et al., EMBO J 3: 2717-2722(1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich etal., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a specific technique fortransformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species.Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain which may depend of thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 forpCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). Thetransfer of the recombinant binary vector to Agrobacterium isaccomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Höfgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacteriumusually involves co-cultivation of the Agrobacterium with explants fromthe plant and follows protocols well known in the art. Transformedtissue is regenerated on selectable medium carrying the antibiotic orherbicide resistance marker present between the binary plasmid T-DNAborders.

Another approach to transforming plant cells with a gene involvespropelling inert or biologically active particles at plant tissues andcells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedureinvolves propelling inert or biologically active particles at the cellsunder conditions effective to penetrate the outer surface of the celland afford incorporation within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the desired gene.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing DNA sought to be introduced) canalso be propelled into plant cell tissue.

2. Transformation of Monocotyledons

Transformation of most monocotyledon species has now also becomeroutine. Specific techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth these techniques are suitable for use with this invention.Co-transformation may have the advantage of avoiding complete vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable. However, a disadvantage of the use of co-transformation isthe less than 100% frequency with which separate DNA species areintegrated into the genome (Schocher et al. Biotechnology 4: 1093-1096(1986)).

Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describetechniques for the preparation of callus and protoplasts from an eliteinbred line of maize, transformation of protoplasts using PEG orelectroporation, and the regeneration of maize plants from transformedprotoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Frommet al. (Biotechnology 8: 833-839 (1990)) have published techniques fortransformation of A188-derived maize line using particle bombardment.Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200(1993)) describe techniques for the transformation of elite inbred linesof maize by particle bombardment. This technique utilizes immature maizeembryos of 1.5-2.5 mm length excised from a maize ear 14-15 days afterpollination and a PDS-1000He Biolistics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfertechniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988);Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology8: 736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).Furthermore, WO 93/21335 describes techniques for the transformation ofrice via electroporation.

Patent Application EP 0 332 581 describes techniques for the generation,transformation and regeneration of Pooideae protoplasts. Thesetechniques allow the transformation of Dactylis and wheat. Furthermore,wheat transformation has been described by Vasil et al. (Biotechnology10: 667-674 (1992)) using particle bombardment into cells of type Clong-term regenerable callus, and also by Vasil et al. (Biotechnology11: 1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084(1993)) using particle bombardment of immature embryos and immatureembryo-derived callus. A specific technique for wheat transformation,however, involves the transformation of wheat by particle bombardment ofimmature embryos and includes either a high sucrose or a high maltosestep prior to gene delivery. Prior to bombardment, any number of embryos(0.75-1 mm in length) are plated onto MS medium with 3% sucrose(Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l2,4-D for induction of somatic embryos, which is allowed to proceed inthe dark. On the chosen day of bombardment, embryos are removed from theinduction medium and placed onto the osmoticum (i.e. induction mediumwith sucrose or maltose added at the desired concentration, typically15%). The embryos are allowed to plasmolyze for 2-3 hours and are thenbombarded. Twenty embryos per target plate is typical, although notcritical. An appropriate gene-carrying plasmid (such as pCIB3064 orpSG35) is precipitated onto micrometer size gold particles usingstandard procedures. Each plate of embryos is shot with the DuPontBiolistics® helium device using a burst pressure of ˜1000 psi using astandard 80 mesh screen. After bombardment, the embryos are placed backinto the dark to recover for about 24 hours (still on osmoticum). After24 hrs, the embryos are removed from the osmoticum and placed back ontoinduction medium where they stay for about a month before regeneration.Approximately one month later the embryo explants with developingembryogenic callus are transferred to regeneration medium (MS+1 mg/literNAA, 5 mg/liter GA), further containing the appropriate selection agent(10 mg/l basta in the case of pCIB3064 and 2 mg/1 methotrexate in thecase of pSOG35). After approximately one month, developed shoots aretransferred to larger sterile containers known as “GA7s” which containhalf-strength MS, 2% sucrose, and the same concentration of selectionagent.

Transformation of monocotyledons using Agrobacterium has also beendescribed. See, WO 94/00977 and U.S. Pat. No. 5,591,616, both of whichare incorporated herein by reference. See also, Negrotto et al., PlantCell Reports 19: 798-803 (2000), incorporated herein by reference. Forthis example, rice (Oryza sativa) is used for generating transgenicplants. Various rice cultivars can be used (Hiei et al., 1994, PlantJournal 6:271-282; Dong et al., 1996, Molecular Breeding 2:267-276; Hieiet al., 1997, Plant Molecular Biology, 35:205-218). Also, the variousmedia constituents described below may be either varied in quantity orsubstituted. Embryogenic responses are initiated and/or cultures areestablished from mature embryos by culturing on MS-CIM medium (MS basalsalts, 4.3 g/liter; B5 vitamins (200×), 5 ml/liter; Sucrose, 30 g/liter;proline, 500 mg/liter; glutamine, 500 mg/liter; casein hydrolysate, 300mg/liter; 2,4-D (1 mg/ml), 2 mil/liter; adjust pH to 5.8 with 1 N KOH;Phytagel, 3 g/liter). Either mature embryos at the initial stages ofculture response or established culture lines are inoculated andco-cultivated with the Agrobacterium tumefaciens strain LBA4404(Agrobacterium) containing the desired vector construction.Agrobacterium is cultured from glycerol stocks on solid YPC medium (100mg/L spectinomycin and any other appropriate antibiotic) for ˜2 days at28° C. Agrobacterium is re-suspended in liquid MS-CIM medium. TheAgrobacterium culture is diluted to an OD600 of 0.2-0.3 andacetosyringone is added to a final concentration of 200 uM.Acetosyringone is added before mixing the solution with the ricecultures to induce Agrobacterium for DNA transfer to the plant cells.For inoculation, the plant cultures are immersed in the bacterialsuspension. The liquid bacterial suspension is removed and theinoculated cultures are placed on co-cultivation medium and incubated at22° C. for two days. The cultures are then transferred to MS-CIM mediumwith Ticarcilin (400 mg/liter) to inhibit the growth of Agrobacterium.For constructs utilizing the PMI selectable marker gene (Reed et al., InVitro Cell. Dev. Biol.-Plant 37:127-132), cultures are transferred toselection medium containing Mannose as a carbohydrate source (MS with 2%Mannose, 300 mg/liter Ticarcillin) after 7 days, and cultured for 3-4weeks in the dark. Resistant colonies are then transferred toregeneration induction medium (MS with no 2,4-D, 0.5 mg/liter IAA, 1mg/liter zeatin, 200 mg/liter timentin 2% Mannose and 3% Sorbitol) andgrown in the dark for 14 days. Proliferating colonies are thentransferred to another round of regeneration induction media and movedto the light growth room. Regenerated shoots are transferred to GA7containers with GA7-1 medium (MS with no hormones and 2% Sorbitol) for 2weeks and then moved to the greenhouse when they are large enough andhave adequate roots. Plants are transplanted to soil in the greenhouse(TO generation) grown to maturity, and the T1 seed is harvested.

3. Transformation of Plastids

Seeds of Nicotiana tabacum c.v. ‘Xanthi nc’ are germinated seven perplate in a 1″ circular array on T agar medium and bombarded 12-14 daysafter sowing with 1 μm tungsten particles (M10, Biorad, Hercules,Calif.) coated with DNA from plasmids pPH143 and pPH145 essentially asdescribed (Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917). Bombardedseedlings are incubated on T medium for two days after which leaves areexcised and placed abaxial side up in bright light (350-500 μmolphotons/m2/s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. andMaliga, P. (1990) PNAS 87, 8526-8530) containing 500 μg/ml spectinomycindihydrochloride (Sigma, St. Louis, Mo.). Resistant shoots appearingunderneath the bleached leaves three to eight weeks after bombardmentare subcloned onto the same selective medium, allowed to form callus,and secondary shoots isolated and subcloned. Complete segregation oftransformed plastid genome copies (homoplasmicity) in independentsubclones is assessed by standard techniques of Southern blotting(Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digestedtotal cellular DNA (Mettler, I. J. (1987) Plant Mol Biol Reporter 5,346-349) is separated on 1% Tris-borate (TBE) agarose gels, transferredto nylon membranes (Amersham) and probed with 32P-labeled random primedDNA sequences corresponding to a 0.7 kb BamHI/HindIII DNA fragment frompCB containing a portion of the rps7/12 plastid targeting sequence.Homoplasmic shoots are rooted aseptically on spectinomycin-containingMS/IBA medium (McBride, K. E. et al. (1994) PNAS 91, 7301-7305) andtransferred to the greenhouse.

V. Breeding and Seed Production

A. Breeding

The plants obtained via transformation with a nucleic acid sequence ofthe present invention can be any of a wide variety of plant species,including those of monocots and dicots; however, the plants used in themethod of the invention are specifically selected from the list ofagronomically important target crops set forth supra. The expression ofa gene of the present invention in combination with othercharacteristics important for production and quality can be incorporatedinto plant lines through breeding. Breeding approaches and techniquesare known in the art. See, for example, Welsh J. R., Fundamentals ofPlant Genetics and Breeding, John Wiley & Sons, NY (1981); CropBreeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wis.(1983); Mayo O., The Theory of Plant Breeding, Second Edition, ClarendonPress, Oxford (1987); Singh, D. P., Breeding for Resistance to Diseasesand Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber,Quantitative Genetics and Selection Plant Breeding, Walter de Gruyterand Co., Berlin (1986).

The genetic properties engineered into the transgenic seeds and plantsdescribed above are passed on by sexual reproduction or vegetativegrowth and can thus be maintained and propagated in progeny plants.Generally said maintenance and propagation make use of knownagricultural methods developed to fit specific purposes such as tilling,sowing or harvesting. Specialized processes such as hydroponics orgreenhouse technologies can also be applied. As the growing crop isvulnerable to attack and damages caused by insects or infections as wellas to competition by weed plants, measures are undertaken to controlweeds, plant diseases, insects, nematodes, and other adverse conditionsto improve yield. These include mechanical measures such a tillage ofthe soil or removal of weeds and infected plants, as well as theapplication of agrochemicals such as herbicides, fungicides,gametocides, nematicides, growth regulants, ripening agents andinsecticides.

Use of the advantageous genetic properties of the transgenic plants andseeds according to the invention can further be made in plant breeding,which aims at the development of plants with improved properties such astolerance of pests, herbicides, or stress, improved nutritional value,increased yield, or improved structure causing less loss from lodging orshattering. The various breeding steps are characterized by well-definedhuman intervention such as selecting the lines to be crossed, directingpollination of the parental lines, or selecting appropriate progenyplants. Depending on the desired properties, different breeding measuresare taken. The relevant techniques are well known in the art and includebut are not limited to hybridization, inbreeding, backcross breeding,multiline breeding, variety blend, interspecific hybridization,aneuploid techniques, etc. Hybridization techniques also include thesterilization of plants to yield male or female sterile plants bymechanical, chemical, or biochemical means. Cross pollination of a malesterile plant with pollen of a different line assures that the genome ofthe male sterile but female fertile plant will uniformly obtainproperties of both parental lines. Thus, the transgenic seeds and plantsaccording to the invention can be used for the breeding of improvedplant lines, that for example, increase the effectiveness ofconventional methods such as herbicide or pesticide treatment or allowone to dispense with said methods due to their modified geneticproperties. Alternatively new crops with improved stress tolerance canbe obtained, which, due to their optimized genetic “equipment”, yieldharvested product of better quality than products that were not able totolerate comparable adverse developmental conditions.

B. Seed Production

In seed production, germination quality and uniformity of seeds areessential product characteristics. As it is difficult to keep a cropfree from other crop and weed seeds, to control seedborne diseases, andto produce seed with good germination, fairly extensive and well-definedseed production practices have been developed by seed producers, who areexperienced in the art of growing, conditioning and marketing of pureseed. Thus, it is common practice for the farmer to buy certified seedmeeting specific quality standards instead of using seed harvested fromhis own crop. Propagation material to be used as seeds is customarilytreated with a protectant coating comprising herbicides, insecticides,fungicides, bactericides, nematicides, molluscicides, or mixturesthereof. Customarily used protectant coatings comprise compounds such ascaptan, carboxin, thiram (TMTD®), methalaxyl (Apron®), andpirimiphos-methyl (Actellic®). If desired, these compounds areformulated together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation to provide protection against damage caused by bacterial,fungal or animal pests. The protectant coatings may be applied byimpregnating propagation material with a liquid formulation or bycoating with a combined wet or dry formulation. Other methods ofapplication are also possible such as treatment directed at the buds orthe fruit.

VI. Alteration of Expression of Nucleic Acid Molecules

The alteration in expression of the nucleic acid molecules of thepresent invention is achieved in one of the following ways:

A. “Sense” Suppression

Alteration of the expression of a nucleotide sequence of the presentinvention, specifically reduction of its expression, is obtained by“sense” suppression (referenced in e.g. Jorgensen et al. (1996) PlantMol. Biol. 31, 957-973). In this case, the entirety or a portion of anucleotide sequence of the present invention is comprised in a DNAmolecule. The DNA molecule is specifically operatively linked to apromoter functional in a cell comprising the target gene, specifically aplant cell, and introduced into the cell, in which the nucleotidesequence is expressible. The nucleotide sequence is inserted in the DNAmolecule in the “sense orientation”, meaning that the coding strand ofthe nucleotide sequence can be transcribed. In a specific embodiment,the nucleotide sequence is fully translatable and all the geneticinformation comprised in the nucleotide sequence, or portion thereof, istranslated into a polypeptide. In another specific embodiment, thenucleotide sequence is partially translatable and a short peptide istranslated. In a specific embodiment, this is achieved by inserting atleast one premature stop codon in the nucleotide sequence, which bringtranslation to a halt. In another more specific embodiment, thenucleotide sequence is transcribed but no translation product is beingmade. This is usually achieved by removing the start codon, e.g. the“ATG”, of the polypeptide encoded by the nucleotide sequence. In afurther specific embodiment, the DNA molecule comprising the nucleotidesequence, or a portion thereof, is stably integrated in the genome ofthe plant cell. In another specific embodiment, the DNA moleculecomprising the nucleotide sequence, or a portion thereof, is comprisedin an extrachromosomally replicating molecule.

In transgenic plants containing one of the DNA molecules describedimmediately above, the expression of the nucleotide sequencecorresponding to the nucleotide sequence comprised in the DNA moleculeis specifically reduced. Specifically, the nucleotide sequence in theDNA molecule is at least 70% identical to the nucleotide sequence theexpression of which is reduced, more specifically it is at least 80%identical, yet more specifically at least 90% identical, yet morespecifically at least 95% identical, yet more specifically at least 99%identical

B. “Anti-Sense” Suppression

In another specific embodiment, the alteration of the expression of anucleotide sequence of the present invention, specifically the reductionof its expression is obtained by “anti-sense” suppression. The entiretyor a portion of a nucleotide sequence of the present invention iscomprised in a DNA molecule. The DNA molecule is specificallyoperatively linked to a promoter functional in a plant cell, andintroduced in a plant cell, in which the nucleotide sequence isexpressible. The nucleotide sequence is inserted in the DNA molecule inthe “anti-sense orientation”, meaning that the reverse complement (alsocalled sometimes non-coding strand) of the nucleotide sequence can betranscribed. In a specific embodiment, the DNA molecule comprising thenucleotide sequence, or a portion thereof, is stably integrated in thegenome of the plant cell. In another specific embodiment the DNAmolecule comprising the nucleotide sequence, or a portion thereof, iscomprised in an extrachromosomally replicating molecule. Severalpublications describing this approach are cited for further illustration(Green, P. J. et al., Ann. Rev. Biochem. 55:569-597 (1986); van derKrol, A. R. et al, Antisense Nuc. Acids & Proteins, pp. 125-141 (1991);Abel, P. P. et al., PNASroc. Natl. Acad. Sci. USA 86:6949-6952 (1989);Ecker, J. R. et al., Proc. Natl. Acad. Sci. USANAS 83:5372-5376 (August1986)).

In transgenic plants containing one of the DNA molecules describedimmediately above, the expression of the nucleotide sequencecorresponding to the nucleotide sequence comprised in the DNA moleculeis specifically reduced. Specifically, the nucleotide sequence in theDNA molecule is at least 70% identical to the nucleotide sequence theexpression of which is reduced, more specifically it is at least 80%identical, yet more specifically at least 90% identical, yet morespecifically at least 95% identical, yet more specifically at least 99%identical.

C. Homologous Recombination

In another specific embodiment, at least one genomic copy correspondingto a nucleotide sequence of the present invention is modified in thegenome of the plant by homologous recombination as further illustratedin Paszkowski et al., EMBO Journal 7:4021-26 (1988). This technique usesthe property of homologous sequences to recognize each other and toexchange nucleotide sequences between each by a process known in the artas homologous recombination. Homologous recombination can occur betweenthe chromosomal copy of a nucleotide sequence in a cell and an incomingcopy of the nucleotide sequence introduced in the cell bytransformation. Specific modifications are thus accurately introduced inthe chromosomal copy of the nucleotide sequence. In one embodiment, theregulatory elements of the nucleotide sequence of the present inventionare modified. Such regulatory elements are easily obtainable byscreening a genomic library using the nucleotide sequence of the presentinvention, or a portion thereof, as a probe. The existing regulatoryelements are replaced by different regulatory elements, thus alteringexpression of the nucleotide sequence, or they are mutated or deleted,thus abolishing the expression of the nucleotide sequence. In anotherembodiment, the nucleotide sequence is modified by deletion of a part ofthe nucleotide sequence or the entire nucleotide sequence, or bymutation. Expression of a mutated polypeptide in a plant cell is alsocontemplated in the present invention. More recent refinements of thistechnique to disrupt endogenous plant genes have been described (Kempinet al., Nature 389:802-803 (1997) and Miao and Lam, Plant J., 7:359-365(1995).

In another specific embodiment, a mutation in the chromosomal copy of anucleotide sequence is introduced by transforming a cell with a chimericoligonucleotide composed of a contiguous stretch of RNA and DNA residuesin a duplex conformation with double hairpin caps on the ends. Anadditional feature of the oligonucleotide is for example the presence of2′-O-methylation at the RNA residues. The RNA/DNA sequence is designedto align with the sequence of a chromosomal copy of a nucleotidesequence of the present invention and to contain the desired nucleotidechange. For example, this technique is further illustrated in U.S. Pat.No. 5,501,967 and Zhu et al. (1999) Proc. Natl. Acad. Sci. USA 96:8768-8773.

D. Ribozymes

In a further embodiment, the RNA coding for a polypeptide of the presentinvention is cleaved by a catalytic RNA, or ribozyme, specific for suchRNA. The ribozyme is expressed in transgenic plants and results inreduced amounts of RNA coding for the polypeptide of the presentinvention in plant cells, thus leading to reduced amounts of polypeptideaccumulated in the cells. This method is further illustrated in U.S.Pat. No. 4,987,071.

E. Dominant-Negative Mutants

In another specific embodiment, the activity of the polypeptide encodedby the nucleotide sequences of this invention is changed. This isachieved by expression of dominant negative mutants of the proteins intransgenic plants, leading to the loss of activity of the endogenousprotein.

F. Aptamers

In a further embodiment, the activity of polypeptide of the presentinvention is inhibited by expressing in transgenic plants nucleic acidligands, so-called aptamers, which specifically bind to the protein.Aptamers are preferentially obtained by the SELEX (Systematic Evolutionof Ligands by Exponential Enrichment) method. In the SELEX method, acandidate mixture of single stranded nucleic acids having regions ofrandomized sequence is contacted with the protein and those nucleicacids having an increased affinity to the target are partitioned fromthe remainder of the candidate mixture. The partitioned nucleic acidsare amplified to yield a ligand enriched mixture. After severaliterations a nucleic acid with optimal affinity to the polypeptide isobtained and is used for expression in transgenic plants. This method isfurther illustrated in U.S. Pat. No. 5,270,163.

G. Zinc Finger Proteins

A zinc finger protein that binds a nucleotide sequence of the presentinvention or to its regulatory region is also used to alter expressionof the nucleotide sequence. Specifically, transcription of thenucleotide sequence is reduced or increased. Zinc finger proteins arefor example described in Beerli et al. (1998) PNAS 95:14628-14633., orin WO 95/19431, WO 98/54311, or WO 96/06166, all incorporated herein byreference in their entirety.

H. dsRNA

Alteration of the expression of a nucleotide sequence of the presentinvention is also obtained by dsRNA interference as described forexample in WO 99/32619, WO 99/53050 or WO 99/61631, all incorporatedherein by reference in their entirety. In another specific embodiment,the alteration of the expression of a nucleotide sequence of the presentinvention, specifically the reduction of its expression, is obtained bydouble-stranded RNA (dsRNA) interference. The entirety or, specificallya portion of a nucleotide sequence of the present invention is comprisedin a DNA molecule. The size of the DNA molecule is specifically from 100to 1000 nucleotides or more; the optimal size to be determinedempirically. Two copies of the identical DNA molecule are linked,separated by a spacer DNA molecule, such that the first and secondcopies are in opposite orientations. In the specific embodiment, thefirst copy of the DNA molecule is in the reverse complement (also knownas the non-coding strand) and the second copy is the coding strand; inthe most specific embodiment, the first copy is the coding strand, andthe second copy is the reverse complement. The size of the spacer DNAmolecule is specifically 200 to 10,000 nucleotides, more specifically400 to 5000 nucleotides and most specifically 600 to 1500 nucleotides inlength. The spacer is specifically a random piece of DNA, morespecifically a random piece of DNA without homology to the targetorganism for dsRNA interference, and most specifically a functionalintron which is effectively spliced by the target organism. The twocopies of the DNA molecule separated by the spacer are operativelylinked to a promoter functional in a plant cell, and introduced in aplant cell, in which the nucleotide sequence is expressible. In aspecific embodiment, the DNA molecule comprising the nucleotidesequence, or a portion thereof, is stably integrated in the genome ofthe plant cell. In another specific embodiment the DNA moleculecomprising the nucleotide sequence, or a portion thereof, is comprisedin an extrachromosomally replicating molecule. Several publicationsdescribing this approach are cited for further illustration (Waterhouseet al. (1998) PNAS 95:13959-13964; Chuang and Meyerowitz (2000) PNAS97:4985-4990; Smith et al. (2000) Nature 407:319-320). Alteration of theexpression of a nucleotide sequence by dsRNA interference is alsodescribed in, for example WO 99/32619, WO 99/53050 or WO 99/61631, allincorporated herein by reference in their entirety.

In transgenic plants containing one of the DNA molecules describedimmediately above, the expression of the nucleotide sequencecorresponding to the nucleotide sequence comprised in the DNA moleculeis specifically reduced. Specifically, the nucleotide sequence in theDNA molecule is at least 70% identical to the nucleotide sequence theexpression of which is reduced, more specifically it is at least 80%identical, yet more specifically at least 90% identical, yet morespecifically at least 95% identical, yet more specifically at least 99%identical.

I. Insertion of a DNA Molecule (Insertional Mutagenesis)

In another specific embodiment, a DNA molecule is inserted into achromosomal copy of a nucleotide sequence of the present invention, orinto a regulatory region thereof. Specifically, such DNA moleculecomprises a transposable element capable of transposition in a plantcell, such as e.g. Ac/Ds, Em/Spm, mutator. Alternatively, the DNAmolecule comprises a T-DNA border of an Agrobacterium T-DNA. The DNAmolecule may also comprise a recombinase or integrase recognition sitewhich can be used to remove part of the DNA molecule from the chromosomeof the plant cell. Methods of insertional mutagenesis using T-DNA,transposons, oligonucleotides or other methods known to those skilled inthe art are also encompassed. Methods of using T-DNA and transposon forinsertional mutagenesis are described in Winkler et al. (1989) MethodsMol. Biol. 82:129-136 and Martienssen (1998) PNAS 95:2021-2026,incorporated herein by reference in their entireties.

J. Deletion Mutagenesis

In yet another embodiment, a mutation of a nucleic acid molecule of thepresent invention is created in the genomic copy of the sequence in thecell or plant by deletion of a portion of the nucleotide sequence orregulator sequence. Methods of deletion mutagenesis are known to thoseskilled in the art. See, for example, Miao et al., (1995) Plant J.7:359.

In yet another embodiment, this deletion is created at random in a largepopulation of plants by chemical mutagenesis or irradiation and a plantwith a deletion in a gene of the present invention is isolated byforward or reverse genetics. Irradiation with fast neutrons or gammarays is known to cause deletion mutations in plants (Silverstone et al,(1998) Plant Cell, 10:155-169; Bruggemann et al., (1996) Plant J.,10:755-760; Redei and Koncz in Methods in Arabidopsis Research, WorldScientific Press (1992), pp. 16-82). Deletion mutations in a gene of thepresent invention can be recovered in a reverse genetics strategy usingPCR with pooled sets of genomic DNAs as has been shown in C. elegans(Liu et al., (1999), Genome Research, 9:859-867.). A forward geneticsstrategy would involve mutagenesis of a line displaying PTGS followed byscreening the M2 progeny for the absence of PTGS. Among these mutantswould be expected to be some that disrupt a gene of the presentinvention. This could be assessed by Southern blot or PCR for a gene ofthe present invention with genomic DNA from these mutants.

K. Overexpression in a Plant Cell

In yet another specific embodiment, a nucleotide sequence of the presentinvention encoding a polypeptide is over-expressed. Examples of nucleicacid molecules and expression cassettes for over-expression of a nucleicacid molecule of the present invention are described above. Methodsknown to those skilled in the art of over-expression of nucleic acidmolecules are also encompassed by the present invention.

In a specific embodiment, the expression of the nucleotide sequence ofthe present invention is altered in every cell of a plant. This is forexample obtained though homologous recombination or by insertion in thechromosome. This is also for example obtained by expressing a sense orantisense RNA, zinc finger protein or ribozyme under the control of apromoter capable of expressing the sense or antisense RNA, zinc fingerprotein or ribozyme in every cell of a plant. Constitutive expression,inducible, tissue-specific or developmentally-regulated expression arealso within the scope of the present invention and result in aconstitutive, inducible, tissue-specific or developmentally-regulatedalteration of the expression of a nucleotide sequence of the presentinvention in the plant cell. Constructs for expression of the sense orantisense RNA, zinc finger protein or ribozyme, or for over-expressionof a nucleotide sequence of the present invention, are prepared andtransformed into a plant cell according to the teachings of the presentinvention, e.g. as described infra.

VII. Polypeptides

The present invention further relates to isolated polypeptidescomprising the amino acid sequence of SEQ ID NO:2. In particular,isolated polypeptides comprising the amino acid sequence of SEQ ID NO:2,and variants having conservative amino acid modifications. One skilledin the art will recognize that individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide or protein sequencewhich alters, adds or deletes a single amino acid or a small percent ofamino acids in the encoded sequence is a “conservative modification”where the modification results in the substitution of an amino acid witha chemically similar amino acid. Conservative modified variants providesimilar biological activity as the unmodified polypeptide. Conservativesubstitution tables listing functionally similar amino acids are knownin the art. See Crighton (1984) Proteins, W.H. Freeman and Company.

In a specific embodiment, a polypeptide having substantial similarity toa polypeptide sequence of SEQ ID NO:2, or exon or domain thereof, is anallelic variant of the polypeptide sequence listed in SEQ ID NO:2. Inanother specific embodiment, a polypeptide having substantial similarityto a polypeptide sequence listed in SEQ ID NO:2, or exon or domainthereof, is a naturally occurring variant of the polypeptide sequencelisted SEQ ID NO:2. In another specific embodiment, a polypeptide havingsubstantial similarity to a polypeptide sequence listed SEQ ID NO:2, orexon or domain thereof, is a polymorphic variant of the polypeptidesequence listed in SEQ ID NO:2.

In an alternate specific embodiment, the sequence having substantialsimilarity contains a deletion or insertion of at least one amino acid.In a more specific embodiment, the deletion or insertion is of less thanabout ten amino acids. In a most specific embodiment, the deletion orinsertion is of less than about three amino acids.

In a specific embodiment, the sequence having substantial similarityencodes a substitution in at least one amino acid.

Embodiments of the present invention also contemplate an isolatedpolypeptide containing a polypeptide sequence including

(a) a polypeptide sequence listed in SEQ ID NO:2, or exon or domainthereof;

(b) a polypeptide sequence having substantial similarity to (a);

(c) a polypeptide sequence encoded by a nucleotide sequence identical toor having substantial similarity to a nucleotide sequence listed in SEQID NO:1, or an exon or domain thereof, or a sequence complementarythereto;

(d) a polypeptide sequence encoded by a nucleotide sequence capable ofhybridizing under medium stringency conditions to a nucleotide sequencelisted in SEQ ID NO:1, or to a sequence complementary thereto; or

(e) a functional fragment of (a), (b), (c) or (d).

In another specific embodiment, the polypeptide having substantialsimilarity is an allelic variant of a polypeptide sequence listed in SEQID NO:2, or a fragment, domain, repeat or chimeras thereof. In anotherspecific embodiment, the isolated nucleic acid includes a plurality ofregions from the polypeptide sequence encoded by a nucleotide sequenceidentical to or having substantial similarity to a nucleotide sequencelisted in SEQ ID NO:1, or fragment or domain thereof, or a sequencecomplementary thereto.

In another specific embodiment, the polypeptide is a polypeptidesequence listed in SEQ ID NO:2. In another specific embodiment, thepolypeptide is a functional fragment or domain. In yet another specificembodiment, the polypeptide is a chimera, where the chimera may includefunctional protein domains, including domains, repeats,post-translational modification sites, or other features. In a morespecific embodiment, the polypeptide is a plant polypeptide. In a morespecific embodiment, the plant is a dicot. In a more specificembodiment, the plant is a gymnosperm. In a more specific embodiment,the plant is a monocot. In a more specific embodiment, the monocot is acereal. In a more specific embodiment, the cereal may be, for example,maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale,einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum, andteosinte. In another specific embodiment, the cereal is rice.

In a specific embodiment, the polypeptide is expressed in a specificlocation or tissue of a plant. In a more specific embodiment, thelocation or tissue is for example, but not limited to, epidermis,vascular tissue, meristem, cambium, cortex or pith. In a most specificembodiment, the location or tissue is leaf or sheath, root, flower, anddeveloping ovule or seed. In a more specific embodiment, the location ortissue may be, for example, epidermis, root, vascular tissue, meristem,cambium, cortex, pith, leaf, and flower. In a more specific embodiment,the location or tissue is a seed.

In a specific embodiment, the polypeptide sequence encoded by anucleotide sequence having substantial similarity to a nucleotidesequence listed in SEQ ID NO:1 or a fragment or domain thereof or asequence complementary thereto, includes a deletion or insertion of atleast one nucleotide. In a more specific embodiment, the deletion orinsertion is of less than about thirty nucleotides. In a most specificembodiment, the deletion or insertion is of less than about fivenucleotides.

In a specific embodiment, the polypeptide sequence encoded by anucleotide sequence having substantial similarity to a nucleotidesequence listed in SEQ ID NO:1, or fragment or domain thereof or asequence complementary thereto, includes a substitution of at least onecodon. In a more specific embodiment, the substitution is conservative.

In a specific embodiment, the polypeptide sequences having substantialsimilarity to the polypeptide sequence listed in SEQ ID NO:2, or afragment, domain, repeat or chimeras thereof includes a deletion orinsertion of at least one amino acid.

The polypeptides of the invention, fragments thereof or variants thereofcan comprise any number of contiguous amino acid residues from apolypeptide of the invention, wherein the number of residues is selectedfrom the group of integers consisting of from 10 to the number ofresidues in a full-length polypeptide of the invention. Specifically,the portion or fragment of the polypeptide is a functional protein. Thepresent invention includes active polypeptides having specific activityof at least 20%, 30%, or 40%, and specifically at least 505, 60%, or70%, and most specifically at least 805, 90% or 95% that of the native(non-synthetic) endogenous polypeptide. Further, the substratespecificity (kcat/Km) is optionally substantially similar to the native(non-synthetic), endogenous polypeptide. Typically the Km will be atleast 30%, 40%, or 50% of the native, endogenous polypeptide; and morespecifically at least 605, 70%, 80%, or 90%. Methods of assaying andquantifying measures of activity and substrate specificity are wellknown to those of skill in the art.

The isolated polypeptides of the present invention will elicitproduction of an antibody specifically reactive to a polypeptide of thepresent invention when presented as an immunogen. Therefore, thepolypeptides of the present invention can be employed as immunogens forconstructing antibodies immunoreactive to a protein of the presentinvention for such purposes, but not limited to, immunoassays or proteinpurification techniques. Immunoassays for determining binding are wellknown to those of skill in the art such as, but not limited to, ELISAsor competitive immunoassays.

Embodiments of the present invention also relate to chimericpolypeptides encoded by the isolated nucleic acid molecules of thepresent disclosure including a chimeric polypeptide containing apolypeptide sequence encoded by an isolated nucleic acid containing anucleotide sequence including:

(a) a nucleotide sequence listed in SEQ ID NO:1, or an exon or domainthereof;

(b) a nucleotide sequence having substantial similarity to (a);

(c) a nucleotide sequence capable of hybridizing to (a);

(d) a nucleotide sequence complementary to (a), (b) or (c); and

(e) a nucleotide sequence which is the reverse complement of (a), (b) or(c); or

(f) a functional fragment thereof.

A polypeptide containing a polypeptide sequence encoded by an isolatednucleic acid containing a nucleotide sequence, its complement, or itsreverse complement, encoding a polypeptide including a polypeptidesequence including:

(a) a polypeptide sequence listed in SEQ ID NO:2, or a domain, repeat orchimeras thereof;

(b) a polypeptide sequence having substantial similarity to (a);

(c) a polypeptide sequence encoded by a nucleotide sequence identical toor having substantial similarity to a nucleotide sequence listed in SEQID NO:1, or an exon or domain thereof or a sequence complementarythereto;

(d) a polypeptide sequence encoded by a nucleotide sequence capable ofhybridizing under medium stringency conditions to a nucleotide sequencelisted in SEQ ID NO:1, or to a sequence complementary thereto; and afunctional fragment of (a), (b), (c) or (d); or

(e) a functional fragment thereof.

The isolated nucleic acid molecules of the present invention are usefulfor expressing a polypeptide of the present invention in a recombinantlyengineered cell such as a bacteria, yeast, insect, mammalian or plantcell. The cells produce the polypeptide in a non-natural condition (e.g.in quantity, composition, location and/or time) because they have beengenetically altered to do so. Those skilled in the art are knowledgeablein the numerous expression systems available for expression of nucleicacids encoding a protein of the present invention, and will not bedescribed in detail below.

Briefly, the expression of isolated nucleic acids encoding a polypeptideof the invention will typically be achieved, for example, by operablylinking the nucleic acid or cDNA to a promoter (constitutive orregulatable) followed by incorporation into an expression vector. Thevectors are suitable for replication and/or integration in eitherprokaryotes or eukaryotes. Commonly used expression vectors comprisetranscription and translation terminators, initiation sequences andpromoters for regulation of the expression of the nucleic acid moleculeencoding the polypeptide. To obtain high levels of expression of thecloned nucleic acid molecule, it is desirable to use expression vectorscomprising a strong promoter to direct transcription, a ribosome bindingsite for translation initiation, and a transcription/translationterminator. One skilled in the art will recognize that modifications maybe made to the polypeptide of the present invention without diminishingits biological activity. Some modifications may be made to facilitatethe cloning, expression or incorporation of the polypeptide of theinvention into a fusion protein. Such modification are well known in theart and include, but are not limited to, a methionine added at the aminoterminus to provide an initiation site, or additional amino acids (e.g.poly Histadine) placed on either terminus to create conveniently locatedpurification sequences Restriction sites or termination codons can alsobe introduced into the vector.

In a specific embodiment, the expression vector includes one or moreelements such as, for example, but not limited to, a promoter-enhancersequence, a selection marker sequence, an origin of replication, anepitope-tag encoding sequence, or an affinity purification-tag encodingsequence. In a more specific embodiment, the promoter-enhancer sequencemay be, for example, the CaMV 35S promoter, the CaMV 19S promoter, thetobacco PR-1a promoter, the ubiquitin promoter, and the phaseolinpromoter. In another embodiment, the promoter is operable in plants, andmore specifically, a constitutive or inducible promoter. In anotherspecific embodiment, the selection marker sequence encodes an antibioticresistance gene. In another specific embodiment, the epitope-tagsequence encodes V5, the peptide Phe-His-His-Thr-Thr, hemagglutinin, orglutathione-S-transferase. In another specific embodiment the affinitypurification-tag sequence encodes a polyamino acid sequence or apolypeptide. In a more specific embodiment, the polyamino acid sequenceis polyhistidine. In a more specific embodiment, the polypeptide ischitin binding domain or glutathione-S-transferase. In a more specificembodiment, the affinity purification-tag sequence comprises an inteinencoding sequence.

Prokaryotic cells may be used a host cells, for example, but not limitedto, Escherichia coli, and other microbial strains known to those in theart. Methods for expressing proteins in prokaryotic cells are well knownto those in the art and can be found in many laboratory manuals such asMolecular Cloning: A Laboratory Manual, by J. Sambrook et al. (1989,Cold Spring Harbor Laboratory Press). A variety of promoters, ribosomebinding sites, and operators to control expression are available tothose skilled in the art, as are selectable markers such as antibioticresistance genes. The type of vector chosen is to allow for optimalgrowth and expression in the selected cell type.

A variety of eukaryotic expression systems are available such as, butnot limited to, yeast, insect cell lines, plant cells and mammaliancells. Expression and synthesis of heterologous proteins in yeast iswell known (see Sherman et al., Methods in Yeast Genetics, Cold SpringHarbor Laboratory Press, 1982). Commonly used yeast strains widely usedfor production of eukaryotic proteins are Saccharomyces cerevisiae andPichia pastoris, and vectors, strains and protocols for expression areavailable from commercial suppliers (e.g., Invitrogen).

Mammalian cell systems may be transfected with expression vectors forproduction of proteins. Many suitable host cell lines are available tothose in the art, such as, but not limited to the HEK293, BHK21 and CHOcells lines. Expression vectors for these cells can include expressioncontrol sequences such as an origin of replication, a promoter, (e.g.,the CMV promoter, a HSV tk promoter or phosphoglycerate kinase (pgk)promoter), an enhancer, and protein processing sites such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscription terminator sequences. Other animal cell lines useful forthe production of proteins are available commercially or fromdepositories such as the American Type Culture Collection.

Expression vectors for expressing proteins in insect cells are usuallyderived from the SF9 baculovirus or other viruses known in the art. Anumber of suitable insect cell lines are available including but notlimited to, mosquito larvae, silkworm, armyworm, moth and Drosophilacell lines.

Methods of transfecting animal and lower eukaryotic cells are known.Numerous methods are used to make eukaryotic cells competent tointroduce DNA such as but not limited to: calcium phosphateprecipitation, fusion of the recipient cell with bacterial protoplastscontaining the DNA, treatment of the recipient cells with liposomescontaining the DNA, DEAE dextrin, electroporation, biolistics, andmicroinjection of the DNA directly into the cells. Transfected cells arecultured using means well known in the art (see, Kuchler, R. J.,Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson andRoss, Inc. 1997).

Once a polypeptide of the present invention is expressed it may beisolated and purified from the cells using methods known to thoseskilled in the art. The purification process may be monitored usingWestern blot techniques or radioimmunoassay or other standardimmunoassay techniques. Protein purification techniques are commonlyknown and used by those in the art (see R. Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York 1982: Deutscher,Guide to Protein Purification, Academic Press (1990). Embodiments of thepresent invention provide a method of producing a recombinant protein inwhich the expression vector includes one or more elements including apromoter-enhancer sequence, a selection marker sequence, an origin ofreplication, an epitope-tag encoding sequence, and an affinitypurification-tag encoding sequence. In one specific embodiment, thenucleic acid construct includes an epitope-tag encoding sequence and theisolating step includes use of an antibody specific for the epitope-tag.In another specific embodiment, the nucleic acid construct contains apolyamino acid encoding sequence and the isolating step includes use ofa resin comprising a polyamino acid binding substance, specificallywhere the polyamino acid is polyhistidine and the polyamino bindingresin is nickel-charged agarose resin. In yet another specificembodiment, the nucleic acid construct contains a polypeptide encodingsequence and the isolating step includes the use of a resin containing apolypeptide binding substance, specifically where the polypeptide is achitin binding domain and the resin contains chitin-sepharose.

The polypeptides of the present invention can be synthesized usingnon-cellular synthetic methods known to those in the art. Techniques forsolid phase synthesis are described by Barany and Mayfield, Solid-PhasePeptide Synthesis, pp. 3-284 in the Peptides: Analysis, Synthesis,Biology, Vol. 2, Special Methods in Peptide Synthesis, Part A;Merrifield, et al., J. Am. Chem. Soc. 85:2149-56 (1963) and Stewart etal., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford,Ill. (1984).

The present invention further provides a method for modifying (i.e.increasing or decreasing) the concentration or composition of thepolypeptides of the invention in a plant or part thereof. Modificationcan be effected by increasing or decreasing the concentration and/or thecomposition (i.e. the ratio of the polypeptides of the presentinvention) in a plant. The method comprised introducing into a plantcell with an expression cassette comprising a nucleic acid molecule ofthe present invention, or an nucleic acid encoding a At5g56860 sequenceas described above to obtain a transformed plant cell or tissue,culturing the transformed plant cell or tissue. The nucleic acidmolecule can be under the regulation of a constitutive or induciblepromoter. The method can further comprise inducing or repressingexpression of a nucleic acid molecule of a sequence in the plant for atime sufficient to modify the concentration and/or composition in theplant or plant part.

A plant or plant part having modified expression of a nucleic acidmolecule of the invention can be analyzed and selected using methodsknown to those skilled in the art such as, but not limited to, Southernblot, DNA sequencing, or PCR analysis using primers specific to thenucleic acid molecule and detecting amplicons produced therefrom.

In general, concentration or composition in increased or decreased by atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to anative control plant, plant part or cell lacking the expressioncassette.

Sugars are central regulators of many vital processes in photosyntheticplants, such as photosynthesis, carbon and nitrogen metabolism and thisregulation is achieved by regulating gene expression, either activate orrepress genes involved. The mechanisms by which sugars control geneexpression are not understood well. This GATA transcription factordisclosed here is involved in regulating sugar sensing and theexpression of the factor itself is influenced by the change of the Nstatus. Increased expression of this gene can produce plants withincreased yield, particularly as the manipulation of sugar signalingpathways can lead to increased photosynthesis and increased nitrogenutilization and alter source-sink relationships in seeds, tubes, rootsand other storage organs.

The invention will be further described by reference to the followingdetailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified.

EXAMPLES

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by J. Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (2001); by T. J. Silhavy, M.L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, New York, John Wiley andSons Inc., (1988), Reiter, et al., Methods in Arabidopsis Research,World Scientific Press (1992), and Schultz et al., Plant MolecularBiology Manual, Kluwer Academic Publishers (1.998).

Experimental Background and Procedures

A. Determining Rice and Maize Growth Conditions Under Limiting NitrogenConditions

In past experiments to study genes involved in nitrate uptake andassimilation, the present inventors and others have utilized growthconditions in which nitrate was generally either present in excess orabsent in its entirety. In the latter case, nitrate is typically addedto plants grown in its absence in order to understand nitrate regulationof these and other genes. While this type of extreme treatment is usefulin defining some aspects of gene regulation, it is not suitable to gaina better understanding of the effect of nitrogen limitation. Theinventors have defined conditions for Arabidopsis in which nitrogenlimits growth. This involved developing a system using Rockwool (Hiraiet al., 1995 Plant Cell Physiol 36, 1331-1339) and defining threeconditions: one where growth is maximal; one where nitrogen limitsgrowth to 70-75% maximal growth levels; one where there is a more severelimitation to 30-35% maximal growth levels. The nitrogen limitation actsas a ‘stress’ with the amount of ‘stress’ easily varied by altering theconcentration of nitrate. The inventors assay the physiological“nitrogen status” by measuring nitrate, chlorophyll (which is often usedas a reflection of nitrogen status under field conditions—see, e.g., FoxR H et al 2001 Agron J. 93, 590-597; Minotti P L et al 1994 Hort Science29, 1497-1550), amino acid levels, and nitrate reductase and glutaminesynthetase activities in order to give a baseline in which to assessstudies on mutant lines.

B. Expression Profiling Experiments on Arabidopsis Plants Under NitrogenLimitation

Transcript expression profiling can be used to test RNA levels of largenumbers of genes at the same time. Large numbers of these types ofexperiments have been done in the past, and if the experimental systemis amenable, these can be used to pinpoint the “expression status” of anorganism under different conditions and to use this information to makehypotheses on what genes and pathways are involved in various processes.The inventors found that the more profound the difference in growthconditions, the larger the differences in transcript profiles betweenthe plants grown under these conditions and the more difficult it was todecipher which changes were most important. The only published wholegenome profiling experiment in this area is one in Arabidopsis where anextreme change in nitrate levels was studied (Wang R et al 2003 PlantPhysiol. 132, 556-67). In the case of nitrogen limitations, theinventors studied the effect of growing plants under chronic nitrogenstress as well as changes in the level of available nitrogen. Theinventors have already determined the impact on growth of differentnitrogen levels in Arabidopsis.

The effect of different nitrogen levels on the transcript profiles wasstudied: where nitrogen does not limit growth. For Arabidopsis theinventors collected 4-week old shoots grown under the different nitrogenregimes. Three different samples were collected (biological triplicates)in order to get statistically significant results. The transcriptprofiling was done using Arabidopsis GeneChip® whole genome array(Affymetrix) to study the transcript levels in Arabidopsis. Thebioinformatic analysis necessary to study the considerable data producedby these experiments was performed. By studying the effect of nitrogenlimitation on the expression patterns, the inventors can pinpoint whichpathways are involved in their response to nutrient stress

Example 1 Cloning and Sequence of At5g56860

Gene predictions were derived from the sequence databases, and used todesign oligonucleotide primers for PCR amplification of eitherfull-length (inclusive of the predicted initiation and stop codons, fortransgenic gene overexpression) or partial (for transgenic geneknockout) cDNA clones from rice first strand cDNA. In some instances,these PCR primers included additional 5′ sequences for Gateway™recombination-based cloning (Invitrogen). PCR amplification was carriedout using the HF Advantage II (Clonetech) or EXPAND (Roche) PCR kitsaccording to the manufacturer's instructions. PCR products were clonedinto pCR2.1-TOPO or pDONR201 according to the manufacturer'sinstructions (Invitrogen).

DNAs from 4-8 independent clones were miniprepped following themanufacturer's instructions. DNA was subjected to sequencing analysisusing the BigDye™ Terminator Kit according to manufacturer'sinstructions (ABI). Sequencing made use of primers designed to bothstrands of the predicted gene. All sequencing data were analyzed andassembled using the Phred/Phrap/Consed software package (University ofWashington) to an error ratio equal to or less than 10-4 at theconsensus sequence level.

Consensus sequences were validated as being intact and the correct genein several ways. Initially, restriction analysis was used to confirm thepresence of cDNA inserts of the expected sizes in individual clones. Forfull-length clones, the coding region was checked for absence ofinterruptions (predicted start and stop codons present, no internal stopcodons), by sequencing of the cDNA insert. For both full-length andpartial cDNA clones, alignment with the gene prediction and BLASTanalysis was used to confirm that the intended target gene wasamplified. All cDNA clones generated by PCR were cloned into custom-madebinary destination vectors using Gateway™ recombination-based cloningper the manufacturer's instructions (Invitrogen) Alternatively, PCRproducts were cloned using conventional restriction enzyme-basedcloning.

Expression Vectors and Transformation of Plants

Binary destination vectors for plant transformation consist of a binarybackbone and a T-DNA portion. The binary backbone contains the sequencesnecessary for selection and growth in Escherichia coli DH-5α(Invitrogen) and Agrobacterium tumefaciens LBA4404, including thebacterial spectinomycin antibiotic resistance aadA gene from E. colitransposon Tn7, origins of replication for E. coli (ColE1) and A.tumefaciens (VS1), and the A. tumefaciens virG gene. The T-DNA portionwas flanked by the right and left border sequences and includes thePositech™ (Syngenta) plant selectable marker and a gene expressioncassette which varies depending on the application. The Positech™ plantselectable marker in this instance consists of a rice ACT1 (actin)promoter driving expression of the PMI (phosphomannose isomerase) gene,followed by the cauliflower mosaic virus transcriptional terminator, andconfers resistance to mannose.

The gene expression cassette portion of the binary destination vectorsvaries depending on the application. In general, the cassette consistsof a promoter designed to express the gene in certain tissues of theplant, followed by cloning sites (in some cases interrupted by a segmentof spacer DNA), and finally by the A. tumefaciens nos 3′ endtranscriptional terminator. The promoters used are designed to expressthe gene of interest in specific target tissues (eg. endosperm: riceRS-4, wheat glutelin, maize ADPgpp or γ-zein, or barley α-thionin; eg.embryo: maize globulin or oleosin; eg. aleurone: barley α-amylase; eg.root: maize MSR1 and MRS3; eg. green tissue: maize PEPC) orconstitutively (eg. maize UBI plus intron), depending on the gene ofinterest. The cloning site contains either unique restriction enzymesites (for conventional cloning) and/or a Gateway™ recombination-basedcloning cassette (Invitrogen), in either the forward or reverseorientation. In gene expression cassettes designed for double-strandedinterfering RNA (dsRNA) production, the cloning site is divided by aspacer region (eg. first intron of the rice SH1 gene). The spacer,permits the cloning of two gene fragments one in the forward and one inthe reverse orientation. Antisense (reverse orientation expression) isanother technology available for silencing genes of interest.

Transformation of the nucleic acid molecules of the present inventioninto plants was performed using methods described above in the DetailedDescription. Test kits for detection of plants containing the nucleicacid molecules of the present invention can be produced using generaltechniques for the production of antibodies for the use in ELISA-typeimmunoassays. Alternatively, kits for PCR-type analysis of plant DNA canbe used to test for the presence of the nucleic acid molecules of thepresent invention.

Results

Loss of the At5g56860 Gene Expression Causes Reduced Chlorophyll Levelin the T-DNA Insertion Line

Plants homozygous for the T-DNA insertion mutation in the At5g56860 genewere identified and screened for any phenotypic change. The leaves ofthe mutant plants were pale green due to a reduced total chlorophylllevel. The At5g56860 gene exhibited a single zinc finger with 18residues in the zinc finger loop (C-X2-C-X18-C-X2-C). The full lengthprotein sequence contains 398 amino acids. The T-DNA insertion(SALK_(—)001778) was close to the end of the second exon, causingdeletion of the GATA domain. The expression of the At5g56860 was notdetected in the mutant by RT-PCR.

To determine if the phenotype change of the SALK_(—)001778 line wascaused by the single gene mutation, the inventors made the construct toover-express the Atg56860 gene and transformed this gene back into themutant line. The phenotype in the mutant was complemented in thetransformants in which the expression of the At5g56860 gene wasdetected, demonstrating that the At5g56860 gene was responsible for thereduced chlorophyll phenotype change in the SALK_(—)001778 mutant line.

The mutant was also back-crossed to wild type and heterozygous plantswere identified by PCR having both the wild type and the insertionallele. These plants were allowed to self propagate and the progenyseeds showed a 3:1 segregation with the mutant phenotype being presentonly in those plants homozygous for the T-DNA insertion in the At5g56860gene, indicating that the mutant phenotype was caused by the singleAt5g56860 gene mutation and that this was recessive to the wild-typeallele.

The expression of the At5g56860 Gene is Tissue-Specific and is Regulatedby the Nitrate Status; Same Trend as the Nitrate Assimilation Genes

In order to determine the expression pattern of the AtSg56860 gene,total RNA from buds, leaves and roots was extracted and cDNA made.RT-PCR was used to determine the expression pattern of this gene anddemonstrated that the At5g56860 gene was expressed in buds and leavesbut not in roots. To determine the expression of this gene duringdevelopment, seedlings germinated on MS medium after 3 days and 5 dayswere harvested and total RNA extracted. The At5g56860 gene was found tobe expressed right after germination.

Since chlorophyll level is often used as a reflection of nitrogen status(29-30—need references), the expression of the gene was studied underdifferent nitrogen conditions to determine if its expression level isinfluenced by nitrogen availability. The baseline expression level ofthe At5g56860 grne was lower when grown in a hydroponic culture withvery low nitrate concentration (0.3 mM) comparing with highconcentration (3 mM). In order to determine whether this gene isregulated by nitrate, the plants were transferred from 1 mM nitrate tothe 3 mM nitrate growth conditions. The expression of the At5g56860 genewas up-regulated by the increased nitrate concentration after 2 hrs,with the level of expression level decreasing after 24 hr although itwas still above the baseline expression level. The same expressionpattern was found for the nia1, nia2, and nir, the first two enzymes innitrate reduction, although the magnitude of fold change was larger fornia1, nia2 and nir than for the At5g56860 gene.

The At5g56860-Less Plant Accumulates Less Total N when N Supply isLimiting but the At5g56860 Seems not to Directly Regulate the Expressionof Nitrate Assimilation Genes

Since At5g56860 expression is influenced by the nitrogen status, theinventors tested if different nitrogen conditions would affect thegrowth of the mutant plant. The mutant and wild type plants were grownon conditions limiting for N when the plants are grown in soil (3 mMnitrate) and the ideal nitrogen condition (10 mM nitrate). Shoots of the4-week-old mutant and the control plants were collected, with biomassand total N being measured. There was no significant change in biomass.However, the total N in the mutant was less than in wild type underlimiting N condition and similar to wild type plants when there wassufficient N supply. The expression of nia1, nia2, and nir was analyzedin the mutant plants using real time PCR which showed that nia1, nia2and nir expression in the mutant was not significantly different fromthat in the wild type plant. If the At5g56860 gene is one of theregulatory genes directly controlling nia1, nia2 and nir expression, thebaseline expression level of nia1, nia2 and nir would be altered in theT-DNA mutant lacking the At5g56860 expression. However, this was not thecase.

The At5g56860 Gene Regulates the Expression of Genes Involved inDifferent Functional Categories

A transcriptional profiling experiment was done to compare the baselinegene expression at a whole genome scale in the mutant and the wild typeplant to see which genes have altered expression in the mutant. Again,wild type plants and the SALK_(—)001778 mutant plants were grown undereither limiting N condition (3 mM nitrate) or sufficient N condition (10mM nitrate) and 4-week-old shoots were collected. RNA were extracted andhybridized to Arabidopsis GeneChip® whole genome array. Genes having atleast 1.5 times lower expression in mutant versus wild-type plants grownunder limiting N condition (3 mM nitrate) and genes having at least 1.5times lower expression in mutant versus wild-type plants when grown atsufficient N concentration (10 mM) were analyzed. A browser-basedfunctional classification program (Provart, N. & Zhu, T. (2003) Currentsin Computational Molecular Biology 271-272) was used to show that thedown-regulated genes include those involved in nitrogen and sulfurmetabolism (At3g44300 and At4g27450), in the regulation of C-compoundsand carbohydrate utilization (At2g18700 and At1g70290), in thebiosynthesis of lipid, secondary metabolism, glycosides (At4g37150,At1g09500, At3g09260), in perception of nutrients and nutritionaladaptation (At5g24160), in nutrients uptake and absorption (At1g03220and At2g37770), in electron transport and membrane-associated energyconservation (At5g24160 and At5g20230). The inventors verified some ofthose genes by quantitative RT-PCR and the correlation between the PCRand microarray data was very high.

The At5g56860 Mutant Plant is More Sensitive to Exogenous Glucose

While the At5g56860 appears to regulate the expression of genesimportant for different biological processes, many of these genes areinvolved in C metabolism, such as the sugar and hexose transporters. Inorder to test whether the growth of the mutant plant would be differentfrom the wild type on different C source, these plants were germinatedon different concentration of glucose and sucrose. The average growth ofthe mutant seedlings were stunted compared with the wild type plants ona 6% glucose medium Seedling phenotypes on plates containing 6% glucoseare well-documented bioassays for sugar sensitivity (Jang, J., Leon, P,Zhou, L. & Sheen, J. (1997) Plant Cell 9, 5-19) and the results showedthat the mutant with no expression of the At5g56860 is hypersensitive to6% glucose.

The At5g56860 Gene Appears to Regulate the Expression of HXK1 but notHKX2 and Also Regulates the Hexose Transporter Gene

The At5g56860 gene affects sensitivity to glucose and thus would beexpected to affect the expression of genes involved in glucosemetabolism. Two types of genes have been shown to affect glucosesensing. The first is hexokinase (HXK) since over-expression of HXK inthis gene leads to a phenotype quite similar to that of the insertionmutation in the At5g56860 GATA transcription factor gene. The second isthe hexose transporter gene which has been shown to be involved in sugarsensing in yeast. There are two Arabidopsis HXK genes which wereidentified by genetic complementation of a yeast hxk1hxk2 double mutant(Jang, J., Leon, P, Zhou, L. & Sheen, J. (1997) Plant Cell 9, 5-19). Theresults showed that the baseline HXK1 expression level was higher in themutant comparing to that in the wild type, but the HXK2 level was notaltered at all in the mutant, suggesting that the At5g56860 is anegative regulator of HXK1 but not HXK2.

The 500 bp upstream sequence of HXK1 and HXK2 were searched and theconsensus GATA binding sequence (T/A)GATA(G/A), or (T/C)TATC(T/A) on thecomplementary strain, was present multiple times in the HXK1 promotersequence, while it was absent from the HXK2 promoter sequence (sequencenot shown). The hexose transporter gene was down-regulated in the mutantline and also had the GATA motif present in its promoter region.Although, the presence of a GATA motif is not definitive indemonstrating the regulation of HXK1 and the hexose transporter by aGATA factor, their presence at least supports the notion that it mightbe directly regulating these genes.

Gain-of-Function Transgenic Plants are Sugar Hyposensitive

Transgenic Arabidopsis plants overexpressing the At5g56860 weregenerated by Agrobacterium-mediated transformation (Bechtold, N., Ellis,J. & Pelletier, G. (1993) C R Acad Sci 316, 1194-1199). Transgenicplants were selected on kanamycin containing medium and T2 lines showeda 3:1 segregation ratio on kanamycin which indicate a single insertionof the transgene. These were selected for self pollination. Transgeniclines of the T3 generation homozygous for the transgene were used forfurther analysis. The expression levels of At5g56860 in the transgeniclines were determined by real-time RT-PCR (data not shown). The seedsfrom over-expressing lines were germinated on 6% glucose plates andthere was little inhibitory affect on growth for the over-expressingtransgenic seedlings, while wild-type plants are severely inhibited,indicating they are much less sensitive to the exogenous glucose. It isinteresting to note that the HXK1 gene is not down regulated in thesetransgenic lines (data not shown) while the hexose transporter gene isstrongly up-regulated in these plants.

Discussion

The At5g56860 is Required for the Sugar Sensing

In the T-DNA mutant with no expression of the At5g56860 gene, themutants were sugar hypersensitive, while the transgenic plantsover-expressing the At5g56860 gene show sugar hyposensitivity,demonstrating that the At5g56860 is involved in the regulation of sugarsensing. The hypersensitivity to 6% glucose has been reported intransgenic Arabidopsis plants overexpressing AtHXK1 and AtHXK2 (Jang,J., Leon, P, Zhou, L. & Sheen, J. (1997) Plant Cell 9, 5-19) and thehyposensitivity to 6% glucose was reported in the transgenic Arabidopsisplants expressing antisense AtHXK1 and AtHXK2 (Jang, J., Leon, P, Zhou,L. & Sheen, J. (1997) Plant Cell 9, 5-19). Transgenic seedlingsoverexpressing AtHXK1 were reported to have reduced chlorophyll contentin their leaves (Jang, J., Leon, P. Zhou, L. & Sheen, J. (1997) PlantCell 9, 5-19; Dai, N., Schaffer, A., Petreikov, M., Shahak, Y., Giller,Y., Ratner, K., Levine, A. & Granot, D. (1999) Plant Cell 11,1253-1266). In the T-DNA mutant with no expression of the At5g56860, theAtHXK1 baseline line expression is higher than the wild type control andthey are sugar hypersensitive. The mutants show a reduced chlorophylllevel. No GATA elements are found in the 500 bp upstream sequence of theHXK2 structural gene. There is one GATA element in the −737 region ofthe HXIQK promoter. However, since tandem repeat or two GATA elementslocated within 30 bp or the core sequence (T/A)GATA(G/A) is required forefficient binding (Chiang, T. Y. & Marzluf, G. A. (1994) Biochemistry33, 576-582; Lin, Y., Hwang, C. F., Brown, J. B. & Cheng, C. L. (1994)Plant Physiol 106, 477-484), it is unlikely that HXK2 is directlyregulated by GATA factors. On the contrary, the 500 bp upstream sequenceof the HXK1 has a number of GATA elements. The level of HXK1 expressionis altered in the At5g56860 mutant line, but not the HXK2 which supportsthe notion that this GATA factor regulates the expression of HXK1.

The lines over-expressing At5g56860 are resistant to the presence ofhigh glucose levels. The over-expression of this GATA factor does notlead to a change in expression of HXK1, but does lead to an increase inexpression of the hexose transporter gene. This gene has been found tobe involved in sugar sensing in yeast.

The At5g56860 Expression Responds to a Change of the N Status

The At5g56860 gene expression is apparently influenced by the N statusof the plant. When the nitrate level is low, the baseline expression ofthe At5g56860 gene is relatively low and when the nitrate level is high,the baseline expression level of the At5g56860 gene is relatively high.When plants are switched from low nitrate condition to higher nitratecondition, the expression of the At5g56860 gene is up-regulated. Thisexpression pattern is the same as the key enzymes in the nitrateassimilation pathways such as NR and NiR, although the latter havehigher rates of induction of expression when exposed to high levels ofnitrate. However, the At5g56860 gene does not directly control nia1,nia2 and nir expression as these are not affected in the mutant line.The expression of the At5g56860 gene does have an impact on nitrogenmetabolism, as loss of the At5g56860 gene resulted in reduced total Naccumulation. It is interesting to note that the At5g56860 gene forms aparalogous relationship with the At4g26150 gene from the phylogenetictree of all 30 Arabidopsis GATA transcription factor genes (Riechmann,J. L., Heard, J., Martin, G., Reuber, L., Jiang, C., Keddie, J., Adam,L., Pineda, O., Ratcliffe, O. J., Samaha, R. R., Creelman, R., Pilgrim,M., Broun, P., Zhang, J. Z., Ghandehari, D., Sherman, B. K. & Yu, G.(2000) Science 290, 2105-2110; Reyes, J. C., Muro-Pastor, M. I. &Florencio, F. J. (2004) Plant physiol. 134, 1718-1732). The At4g26150has already been shown to be inducible by nitrate (Wang, R., Okamoto,M., Xing, X. & Crawford, N. M. (2003) Plant Physiol 132, 556-567;Scheible, W., Morcuende, R., Czechowski, T., Fritz, C., Osuna, D.,Palacios-Rojas, D., Schindelasch, D., Thimm, O., Udvardi, M. K. & Stitt,M. (2004) Plant Physiol 136, 2483-2499). However, their function doesnot seem to be redundant as the expression signatures are not completelythe same for the two genes (Czechowski, T., Bari, R., Stitt, M.,Scheible, W. & Udvardi, M. (2004) Plant J. 38, 366-379). In theSALK_(—)001778 mutant where At5g56860 gene expression is lost, theexpression of the At4g26150 gene is increased but this did notcomplement the mutant phenotype.

Crosstalk Between the C and N Regulation

It is a challenge to understand how the C and N signaling pathwaysinteract with each other, not to mention their interaction with othersignaling pathways such as those involved with hormones, light andstress. To the knowledge of the present inventors, this is the firsttrans-acting factor reported to be involved in regulating sugar sensingand it is important to note that the expression of the factor itself isinfluenced by the change of the N status.

Example 2

The full length At5g56860 cDNA (GNC) was amplified from Arabidopsis leafcDNA using the primers 5′-GCTCTAGATTTCTCTCTCTCTTTGTGTCTTCATTTG-3′ (SEQID NO:4) and 5′-gcgagctctcgggtgactaatgttcgttcc-3′ (SEQ ID NO:5). Theresulting ˜1500 bp fragment was verified by sequencing and then digestedby XbaI and SacI and cloned into the XbaI-SacI-digested expressionvector pROK2 containing the cauliflower mosaic virus (CaMV) 35S promoterdriving the GNC expression in a constitutive, high-level fashion (FIG.4). The p35S-GNC binary vector was transformed into Agrobacteriumtumefaciens strain EHA105 and the resulting Agrobacterium strain wasused to transform the wild type plants (Col-0) and the transformantswere selected on kanamycin resistance. Plants with a single insertion ofthe transgene were selected for self pollination to generate T3homozygous lines for further analysis. Over-expression of GNC in thetransgenics was confirmed by quantitative RT-PCR. Wild type plants andGNC over-expression plants were grown under sufficient nitrogen (10 mMnitrate) and limiting nitrogen (3 mM nitrate) conditions. No obviousdifference was observed in the initial growth stages for both nitrogenconditions. After ˜28 days when plants started entering the reproductivestage, leaf senescence was observed in the wild type plants grown underlimiting nitrogen condition but not in the GNC over-expression plantsand this difference in senescence was clearly observable in the periodafter this stage. In addition, total chlorophyll level was ˜15% higherin those GNC over-expression plants. The chlorophyll level is anindicator of nitrogen availability and photosynthetic capacityindicating that these over-expressing lines are healthier under areduced nitrogen regime. Neither wild type nor GNC over-expressionplants of 4-week-old showed these differences under the sufficientnitrogen condition. There was no significant difference in chlorophylllevel between wild type and GNC over-expression plants under sufficientnitrogen condition.

Having now described particular embodiments of the invention by way ofthe foregoing examples, which are not intended to be limiting, theinvention will now be further set forth in the following claims. Thoseskilled in the art will recognize that the claims also permit for theinclusion of equivalents beyond the claims' literal scope.

What is claimed is:
 1. A method of producing a transgenic plantcomprising: (a) transforming a plant cell with a nucleic acid moleculecomprising SEQ ID NO:3; and (b) growing the plant cell into a plant thatexpresses SEQ ID NO:3 wherein expressing the nucleic acid moleculeresults in an elevated level of expression of SEQ ID NO:3 as compared toa plant that has not been transformed with SEQ ID NO:3; and (c)selecting a transgenic plant having improved or increased nitrogenutilization, carbon utilization, seed yield, or chlorophyll levels. 2.The method according to claim 1, wherein the plant is a dicot,gymnosperm, or a monocot.
 3. The method according to claim 2, whereinthe monocot is selected from the group consisting of maize, wheat,barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt,emmer, teff, milo, flax, gramma grass, Tripsacum sp., and teosinte. 4.The method according to claim 2, wherein the dicot is selected from thegroup consisting of soybean, tobacco, or cotton.
 5. The method accordingto claim 1, wherein the nucleic acid molecule is expressed in a specificlocation or tissue of the plant.
 6. The method according to claim 5,wherein the location or tissue is selected from one or more of seed,epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf,and flower.
 7. The method according to claim 6, wherein the location ortissue is a seed.
 8. The method according to claim 1, wherein thenucleic acid molecule is in an expression cassette comprising a promotersequence operably linked to the nucleic acid molecule.
 9. A method ofproducing a transgenic plant comprising: (a) transforming a plant cellwith a nucleic acid molecule comprising a nucleic acid sequence shown inSEQ ID NO: 3 or a sequence that hybridizes to SEQ ID NO: 3 understringent hybridization conditions comprising 7% sodium dodecyl sulfate(SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.2×SSC at 65°C., wherein the sequence that hybridizes encodes a functional GATAtranscription factor; (b) growing the plant cell into a plant thatexpresses SEQ ID NO:3 wherein expressing the nucleic acid moleculeresults in an elevated level of expression of SEQ ID NO:3 as compared toa plant that has not been transformed with SEQ ID NO:3; and (c)selecting a transgenic plant having improved or increased nitrogenutilization, carbon utilization, seed yield, or chlorophyll levels. 10.The method according to claim 9, wherein the plant is a dicot,gymnosperm, or a monocot.
 11. The method according to claim 10, whereinthe monocot is selected from the group consisting of maize, wheat,barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt,emmer, teff, milo, flax, gramma grass, Tripsacum sp., and teosinte. 12.The method according to claim 10, wherein the dicot is selected from thegroup consisting of soybean, tobacco, or cotton.
 13. The methodaccording to claim 9, wherein the nucleic acid molecule is expressed ina specific location or tissue of the plant.
 14. The method according toclaim 13, wherein the location or tissue is selected from one or more ofseed, epidermis, root, vascular tissue, meristem, cambium, cortex, pith,leaf, and flower.
 15. The method according to claim 14, wherein thelocation or tissue is a seed.
 16. The method according to claim 9,wherein the nucleic acid molecule is in an expression cassettecomprising a promoter sequence operably linked to the nucleic acidmolecule.